> top > projects > sentences > docs > PMC:3320587 > annotations
sentences Home  

PMC:3320587 JSONTXT 30 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T57 0-133 Sentence denotes Regulation of Mycobacterium tuberculosis-Dependent HIV-1 Transcription Reveals a New Role for NFAT5 in the Toll-Like Receptor Pathway
T1 0-133 Sentence denotes Regulation of Mycobacterium tuberculosis-Dependent HIV-1 Transcription Reveals a New Role for NFAT5 in the Toll-Like Receptor Pathway
T2 134-183 Sentence denotes NFAT5 Modulation of MTb-Induced HIV-1 Replication
T3 185-193 Sentence denotes Abstract
T58 194-414 Sentence denotes Tuberculosis (TB) disease in HIV co-infected patients contributes to increased mortality by activating innate and adaptive immune signaling cascades that stimulate HIV-1 replication, leading to an increase in viral load.
T4 194-414 Sentence denotes Tuberculosis (TB) disease in HIV co-infected patients contributes to increased mortality by activating innate and adaptive immune signaling cascades that stimulate HIV-1 replication, leading to an increase in viral load.
T59 415-668 Sentence denotes Here, we demonstrate that silencing of the expression of the transcription factor nuclear factor of activated T cells 5 (NFAT5) by RNA interference (RNAi) inhibits Mycobacterium tuberculosis (MTb)-stimulated HIV-1 replication in co-infected macrophages.
T5 415-668 Sentence denotes Here, we demonstrate that silencing of the expression of the transcription factor nuclear factor of activated T cells 5 (NFAT5) by RNA interference (RNAi) inhibits Mycobacterium tuberculosis (MTb)-stimulated HIV-1 replication in co-infected macrophages.
T60 669-972 Sentence denotes We show that NFAT5 gene and protein expression are strongly induced by MTb, which is a Toll-like receptor (TLR) ligand, and that an intact NFAT5 binding site in the viral promoter of R5-tropic HIV-1 subtype B and subtype C molecular clones is required for efficent induction of HIV-1 replication by MTb.
T6 669-972 Sentence denotes We show that NFAT5 gene and protein expression are strongly induced by MTb, which is a Toll-like receptor (TLR) ligand, and that an intact NFAT5 binding site in the viral promoter of R5-tropic HIV-1 subtype B and subtype C molecular clones is required for efficent induction of HIV-1 replication by MTb.
T61 973-1188 Sentence denotes Furthermore, silencing by RNAi of key components of the TLR pathway in human monocytes, including the downstream signaling molecules MyD88, IRAK1, and TRAF6, significantly inhibits MTb-induced NFAT5 gene expression.
T7 973-1188 Sentence denotes Furthermore, silencing by RNAi of key components of the TLR pathway in human monocytes, including the downstream signaling molecules MyD88, IRAK1, and TRAF6, significantly inhibits MTb-induced NFAT5 gene expression.
T62 1189-1401 Sentence denotes Thus, the innate immune response to MTb infection induces NFAT5 gene and protein expression, and NFAT5 plays a crucial role in MTb regulation of HIV-1 replication via a direct interaction with the viral promoter.
T8 1189-1401 Sentence denotes Thus, the innate immune response to MTb infection induces NFAT5 gene and protein expression, and NFAT5 plays a crucial role in MTb regulation of HIV-1 replication via a direct interaction with the viral promoter.
T63 1402-1511 Sentence denotes These findings also demonstrate a general role for NFAT5 in TLR- and MTb-mediated control of gene expression.
T9 1402-1511 Sentence denotes These findings also demonstrate a general role for NFAT5 in TLR- and MTb-mediated control of gene expression.
T10 1513-1527 Sentence denotes Author Summary
T11 1528-1662 Sentence denotes The major cause of AIDS deaths globally has been tuberculosis (TB), which is caused by the bacterium Mycobacterium tuberculosis (MTb).
T12 1663-1844 Sentence denotes Co-infection with MTb exacerbates human immunodeficiency virus type1 (HIV-1) replication and disease progression via both innate and adaptive host immune responses to MTb infection.
T13 1845-2017 Sentence denotes In this report, we present evidence that the transcription factor NFAT5 plays a crucial role in MTb-induced HIV-1 replication in human peripheral blood cells and monocytes.
T14 2018-2238 Sentence denotes We also show that MTb infection itself stimulates NFAT5 gene expression in human monocytes and that its expression involves the TLR signalling pathway and requires the downstream adaptor proteins MyD88, IRAK1, and TRAF6.
T15 2239-2486 Sentence denotes This identification of a novel role for NFAT5 in TB/HIV-1 co-infection reveals that NFAT5 is a major mediator of TLR-dependent gene expression and thus provides a potential new therapeutic target for treatment of HIV-1 and possibly other diseases.
T16 2488-2500 Sentence denotes Introduction
T925 2501-2707 Sentence denotes Mycobacterium tuberculosis (MTb), the causative agent of tuberculosis (TB), is the most common co-infection and cause of death in patients infected with human immunodeficiency virus type 1 (HIV-1) [1], [2].
T17 2501-2707 Sentence denotes Mycobacterium tuberculosis (MTb), the causative agent of tuberculosis (TB), is the most common co-infection and cause of death in patients infected with human immunodeficiency virus type 1 (HIV-1) [1], [2].
T926 2708-2904 Sentence denotes Direct engagement of pathogen recognition receptors (PRRs) by MTb on mononuclear phagocytes activates signaling cascades that directly induce transcription from the proviral LTR (reviewed in [3]).
T18 2708-2904 Sentence denotes Direct engagement of pathogen recognition receptors (PRRs) by MTb on mononuclear phagocytes activates signaling cascades that directly induce transcription from the proviral LTR (reviewed in [3]).
T927 2905-3157 Sentence denotes Furthermore, inflammatory cytokines and chemokines produced by the human host in response to MTb infection activate signal transduction pathways in CD4 T cells and monocytic cells that also result in transcriptional activation of the HIV-1 LTR [4]–[6].
T19 2905-3157 Sentence denotes Furthermore, inflammatory cytokines and chemokines produced by the human host in response to MTb infection activate signal transduction pathways in CD4 T cells and monocytic cells that also result in transcriptional activation of the HIV-1 LTR [4]–[6].
T928 3158-3352 Sentence denotes Activation of HIV-1 replication via these MTb-induced pathways ultimately leads to higher viral loads and, in turn, expedited CD4 T cell loss and progression to AIDS ([7], reviewed in [8]–[10]).
T20 3158-3352 Sentence denotes Activation of HIV-1 replication via these MTb-induced pathways ultimately leads to higher viral loads and, in turn, expedited CD4 T cell loss and progression to AIDS ([7], reviewed in [8]–[10]).
T929 3353-3573 Sentence denotes Furthermore, the progressive immune compromise associated with HIV-1 infection itself is a major cause of latent MTb reactivation, as well as increased susceptibility to primary TB infection ([11]–[15], reviewed in [8]).
T21 3353-3573 Sentence denotes Furthermore, the progressive immune compromise associated with HIV-1 infection itself is a major cause of latent MTb reactivation, as well as increased susceptibility to primary TB infection ([11]–[15], reviewed in [8]).
T930 3574-3676 Sentence denotes The primary PRR on monocytic cells triggered by MTb infection is toll-like receptor (TLR) 2 [16]–[20].
T22 3574-3676 Sentence denotes The primary PRR on monocytic cells triggered by MTb infection is toll-like receptor (TLR) 2 [16]–[20].
T931 3677-3892 Sentence denotes Engagement of TLR2 results in engagement of the adaptor protein MyD88 and the subsequent recruitment of several kinases, including IRAK1 and IRAK4, and the ubiquitin ligase TRAF6 ([21]–[23], reviewed in [10], [24]).
T23 3677-3892 Sentence denotes Engagement of TLR2 results in engagement of the adaptor protein MyD88 and the subsequent recruitment of several kinases, including IRAK1 and IRAK4, and the ubiquitin ligase TRAF6 ([21]–[23], reviewed in [10], [24]).
T932 3893-4217 Sentence denotes TRAF6 activates IκB kinase (IKK) and mitogen-activated protein (MAP) kinases that, in turn, ultimately induce activation of specific transcription factor families, including the NF-κB and AP-1 families, which have been shown to associate with the HIV-1 LTR and to drive its transcription ([22], [25]–[27], reviewed in [10]).
T24 3893-4217 Sentence denotes TRAF6 activates IκB kinase (IKK) and mitogen-activated protein (MAP) kinases that, in turn, ultimately induce activation of specific transcription factor families, including the NF-κB and AP-1 families, which have been shown to associate with the HIV-1 LTR and to drive its transcription ([22], [25]–[27], reviewed in [10]).
T933 4218-4370 Sentence denotes Notably, HIV-1 comprises several subtypes, and the LTR of each subtype is unique with respect to the number and organization of activator binding sites.
T25 4218-4370 Sentence denotes Notably, HIV-1 comprises several subtypes, and the LTR of each subtype is unique with respect to the number and organization of activator binding sites.
T934 4371-4566 Sentence denotes For example, HIV-1 subtype B, the most highly characterized viral subtype and the primary cause of infection in the Americas, Europe, Japan, and Australia, has two tandem NF-κB motifs in its LTR.
T26 4371-4566 Sentence denotes For example, HIV-1 subtype B, the most highly characterized viral subtype and the primary cause of infection in the Americas, Europe, Japan, and Australia, has two tandem NF-κB motifs in its LTR.
T935 4567-4763 Sentence denotes By contrast, HIV-1 subtypes C and E, which have spread disproportionately in TB-burdened sub-Saharan Africa and southeast Asia, have three and one NF-κB binding sites, respectively [1], [28]–[30].
T27 4567-4763 Sentence denotes By contrast, HIV-1 subtypes C and E, which have spread disproportionately in TB-burdened sub-Saharan Africa and southeast Asia, have three and one NF-κB binding sites, respectively [1], [28]–[30].
T936 4764-5038 Sentence denotes We previously showed that the most primordial member of the nuclear factor of activated T cells (NFAT) family, NFAT5 (also known as TonEBP), binds to a site within the HIV-1 LTR that is highly conserved across all HIV-1 subtypes, and is also conserved in HIV-2 and SIV LTRs.
T28 4764-5038 Sentence denotes We previously showed that the most primordial member of the nuclear factor of activated T cells (NFAT) family, NFAT5 (also known as TonEBP), binds to a site within the HIV-1 LTR that is highly conserved across all HIV-1 subtypes, and is also conserved in HIV-2 and SIV LTRs.
T937 5039-5260 Sentence denotes This NFAT5 site overlaps the core NF-κB binding motifs in the LTR and is required for constitutive replication of representative HIV-1 subtype B, C, and E isolates in human primary monocyte-derived macrophages (MDM) [31].
T29 5039-5260 Sentence denotes This NFAT5 site overlaps the core NF-κB binding motifs in the LTR and is required for constitutive replication of representative HIV-1 subtype B, C, and E isolates in human primary monocyte-derived macrophages (MDM) [31].
T938 5261-5586 Sentence denotes Given that NFAT5 has previously been shown to be transcriptionally activated by the MAP kinase p38, which is downstream of MyD88 signaling, [32], we speculated that NFAT5 may also be involved in MTb-induced activation of HIV-1 replication via a TLR-mediated pathway in monocytes and peripheral blood mononuclear cells (PBMC).
T30 5261-5586 Sentence denotes Given that NFAT5 has previously been shown to be transcriptionally activated by the MAP kinase p38, which is downstream of MyD88 signaling, [32], we speculated that NFAT5 may also be involved in MTb-induced activation of HIV-1 replication via a TLR-mediated pathway in monocytes and peripheral blood mononuclear cells (PBMC).
T939 5587-5748 Sentence denotes Here, we show that NFAT5 and its cognate binding site are of crucial importance for efficient MTb-induced stimulation of HIV-1 replication in human MDM and PBMC.
T31 5587-5748 Sentence denotes Here, we show that NFAT5 and its cognate binding site are of crucial importance for efficient MTb-induced stimulation of HIV-1 replication in human MDM and PBMC.
T940 5749-5872 Sentence denotes Moreover, we demonstrate that MTb infection increases NFAT5 gene expression in human monocytes in a MyD88-dependent manner.
T32 5749-5872 Sentence denotes Moreover, we demonstrate that MTb infection increases NFAT5 gene expression in human monocytes in a MyD88-dependent manner.
T941 5873-6081 Sentence denotes Thus, these results expand the known stimuli of NFAT5 expression to the PRR-mediated innate immune response, and demonstrate that NFAT5 is a critical modulator of MTb-induced enhancement of HIV-1 replication.
T33 5873-6081 Sentence denotes Thus, these results expand the known stimuli of NFAT5 expression to the PRR-mediated innate immune response, and demonstrate that NFAT5 is a critical modulator of MTb-induced enhancement of HIV-1 replication.
T34 6083-6104 Sentence denotes Materials and Methods
T2810 6106-6122 Sentence denotes Ethics statement
T35 6106-6122 Sentence denotes Ethics statement
T2811 6123-6302 Sentence denotes In our studies we used unidentified human discarded blood cells (peripheral blood mononuclear cells, PBMC), which we obtained from the Blood Bank of Children's Hospital in Boston.
T36 6123-6302 Sentence denotes In our studies we used unidentified human discarded blood cells (peripheral blood mononuclear cells, PBMC), which we obtained from the Blood Bank of Children's Hospital in Boston.
T2904 6304-6316 Sentence denotes Cell culture
T37 6304-6316 Sentence denotes Cell culture
T2905 6317-6654 Sentence denotes PBMC from normal unidentified donors were isolated by Ficoll-Hypaque (Pharmacia Corporation, Peapack, NJ) density gradient centrifugation and were cultured in RPMI 1640 medium with 2 mM L-glutamine (BioWhittaker, Inc., Walkersville, MD) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gemini Bio-Products, www.gembio.com).
T38 6317-6654 Sentence denotes PBMC from normal unidentified donors were isolated by Ficoll-Hypaque (Pharmacia Corporation, Peapack, NJ) density gradient centrifugation and were cultured in RPMI 1640 medium with 2 mM L-glutamine (BioWhittaker, Inc., Walkersville, MD) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gemini Bio-Products, www.gembio.com).
T2906 6655-7081 Sentence denotes Human monocytes were isolated from PBMC preparations by positive selection with CD14 microbeads from Miltenyi Biotec (www.miltenyibiotec.com) as described by the manufacturer, and were cultured at 1×106 cells per well in 6-well plates in Macrophage-SFM medium (Gibco, www.invitrogen.com) supplemented with 15 ng/ml recombinant human MCSF (R&D, www.rndsystems.com) and 5% heat-inactivated human AB serum (Nabi, Boca Raton, FL).
T39 6655-7081 Sentence denotes Human monocytes were isolated from PBMC preparations by positive selection with CD14 microbeads from Miltenyi Biotec (www.miltenyibiotec.com) as described by the manufacturer, and were cultured at 1×106 cells per well in 6-well plates in Macrophage-SFM medium (Gibco, www.invitrogen.com) supplemented with 15 ng/ml recombinant human MCSF (R&D, www.rndsystems.com) and 5% heat-inactivated human AB serum (Nabi, Boca Raton, FL).
T2907 7082-7234 Sentence denotes The cell cultures were incubated at 37°C and 5% CO2 for 5 days, after which supernatant was replaced with fresh medium lacking MCSF before manipulation.
T40 7082-7234 Sentence denotes The cell cultures were incubated at 37°C and 5% CO2 for 5 days, after which supernatant was replaced with fresh medium lacking MCSF before manipulation.
T2908 7235-7353 Sentence denotes More than 95% of the adherent cells obtained with this technique were CD14+ macrophages as verified by flow cytometry.
T41 7235-7353 Sentence denotes More than 95% of the adherent cells obtained with this technique were CD14+ macrophages as verified by flow cytometry.
T2909 7354-7497 Sentence denotes THP-1 cells were obtained from ATCC (www.atcc.org) and cultured in RPMI 1640 medium supplemented with 10% FCS (BioWhittaker, www.lonzabio.com).
T42 7354-7497 Sentence denotes THP-1 cells were obtained from ATCC (www.atcc.org) and cultured in RPMI 1640 medium supplemented with 10% FCS (BioWhittaker, www.lonzabio.com).
T2910 7498-7667 Sentence denotes 293T cells were obtained from ATCC (www.atcc.org) and were maintained in Dulbecco's Modified Eagle's medium (DMEM) (Gibco, www.invitrogen.com) supplemented with 10% FCS.
T43 7498-7667 Sentence denotes 293T cells were obtained from ATCC (www.atcc.org) and were maintained in Dulbecco's Modified Eagle's medium (DMEM) (Gibco, www.invitrogen.com) supplemented with 10% FCS.
T3466 7669-7676 Sentence denotes Viruses
T44 7669-7676 Sentence denotes Viruses
T45 7677-7907 Sentence denotes HIV-1Bal, HIV-1Lai, HIV-193TH64, HIV-192TH51, HIV-192TH53, HIV-198CH01, and HIV-198IN22 were obtained from The Centralized Facility for AIDS Reagents, National Institute for Biological Standard and Control (NIBSC), United Kingdom.
T3467 7677-7974 Sentence denotes HIV-1Bal, HIV-1Lai, HIV-193TH64, HIV-192TH51, HIV-192TH53, HIV-198CH01, and HIV-198IN22 were obtained from The Centralized Facility for AIDS Reagents, National Institute for Biological Standard and Control (NIBSC), United Kingdom. HIV-1KR25 was isolated in our laboratory as described before [33].
T46 7908-7974 Sentence denotes HIV-1KR25 was isolated in our laboratory as described before [33].
T3585 7976-8019 Sentence denotes LTR plasmid construction and reporter assay
T47 7976-8019 Sentence denotes LTR plasmid construction and reporter assay
T3586 8020-8424 Sentence denotes LTR reporter plasmids were constructed by inserting nucleotides −208 to +64 relative to the transcriptional initiation site of HIV-1Lai, HIV-1Bal (B subtype), HIV-198IN17, HIV-198IN22, HIV-198CH01, HIV-1CM9 (C subtype), HIV-193TH64, HIV-192TH53, HIV-192TH51, and HIV-1KR25 (E subtype) into the reporter vector pGL3 (Promega BioSciences, www.promega.com) using Xho I and Hind III restriction enzyme sites.
T48 8020-8424 Sentence denotes LTR reporter plasmids were constructed by inserting nucleotides −208 to +64 relative to the transcriptional initiation site of HIV-1Lai, HIV-1Bal (B subtype), HIV-198IN17, HIV-198IN22, HIV-198CH01, HIV-1CM9 (C subtype), HIV-193TH64, HIV-192TH53, HIV-192TH51, and HIV-1KR25 (E subtype) into the reporter vector pGL3 (Promega BioSciences, www.promega.com) using Xho I and Hind III restriction enzyme sites.
T3587 8425-8502 Sentence denotes Sequences were aligned and analyzed with CLUSTAL W (www.ebi.ac.uk/clustalw/).
T49 8425-8502 Sentence denotes Sequences were aligned and analyzed with CLUSTAL W (www.ebi.ac.uk/clustalw/).
T3588 8503-8627 Sentence denotes The HIV-1Lai NFAT5 binding site-mutant (N5-Mut) reporter plasmid was created by standard PCR-based mutagenesis methods [34].
T50 8503-8627 Sentence denotes The HIV-1Lai NFAT5 binding site-mutant (N5-Mut) reporter plasmid was created by standard PCR-based mutagenesis methods [34].
T3589 8628-8869 Sentence denotes THP-1 cells (0.8×106/ml) were transfected with 0.3 µg/ml LTR wild-type (WT) or mutated reporter plasmids in combination with 0.03 µg/ml Renilla luciferase (pRL-TK) control vector using Effectene transfection reagent (Qiagen; www.qiagen.com).
T51 8628-8869 Sentence denotes THP-1 cells (0.8×106/ml) were transfected with 0.3 µg/ml LTR wild-type (WT) or mutated reporter plasmids in combination with 0.03 µg/ml Renilla luciferase (pRL-TK) control vector using Effectene transfection reagent (Qiagen; www.qiagen.com).
T3590 8870-9011 Sentence denotes Cells were incubated at 37°C for 16 hours after which they were stimulated with 10 µg/ml MTb CDC1551 lysate or left unstimulated for 8 hours.
T52 8870-9011 Sentence denotes Cells were incubated at 37°C for 16 hours after which they were stimulated with 10 µg/ml MTb CDC1551 lysate or left unstimulated for 8 hours.
T3591 9012-9155 Sentence denotes Reporter gene expression was quantitated by dual-luciferase reporter assay according to the manufacturer's protocol (Promega; www.promega.com).
T53 9012-9155 Sentence denotes Reporter gene expression was quantitated by dual-luciferase reporter assay according to the manufacturer's protocol (Promega; www.promega.com).
T4080 9157-9190 Sentence denotes Quantitative DNase I footprinting
T54 9157-9190 Sentence denotes Quantitative DNase I footprinting
T4081 9191-9402 Sentence denotes Recombinant NFAT5 (amino acids 175–471) with an N-terminal 6× His tag was expressed in E. coli BL21(DE3) cells (Stratagene; www.stratagene.com) and purified under native conditions using Ni-NTA agarose (Qiagen).
T55 9191-9402 Sentence denotes Recombinant NFAT5 (amino acids 175–471) with an N-terminal 6× His tag was expressed in E. coli BL21(DE3) cells (Stratagene; www.stratagene.com) and purified under native conditions using Ni-NTA agarose (Qiagen).
T4082 9403-9478 Sentence denotes Recombinant p50 and p65 were purchased (Active Motif, www.activemotif.com).
T56 9403-9478 Sentence denotes Recombinant p50 and p65 were purchased (Active Motif, www.activemotif.com).
T4083 9479-9556 Sentence denotes Quantitative DNase I footprinting was performed as previously described [31].
T57 9479-9556 Sentence denotes Quantitative DNase I footprinting was performed as previously described [31].
T4319 9558-9591 Sentence denotes HIV-1 infectious molecular clones
T58 9558-9591 Sentence denotes HIV-1 infectious molecular clones
T59 9592-9729 Sentence denotes The plasmid encoding the full-length infectious molecular clone of HIV-1Lai was obtained from the NIH AIDS Reagent and Reference Program.
T4320 9592-9989 Sentence denotes The plasmid encoding the full-length infectious molecular clone of HIV-1Lai was obtained from the NIH AIDS Reagent and Reference Program. The HIV-1Lai/Bal-Env infectious molecular clone was constructed by replacing the envelope (env) gp160 amino acids 103–717 of the HIV-1Lai (B subtype that utilizes CXCR4) molecular clone with the corresponding region of HIV-1Bal (B subtype that utilizes CCR5).
T60 9730-9989 Sentence denotes The HIV-1Lai/Bal-Env infectious molecular clone was constructed by replacing the envelope (env) gp160 amino acids 103–717 of the HIV-1Lai (B subtype that utilizes CXCR4) molecular clone with the corresponding region of HIV-1Bal (B subtype that utilizes CCR5).
T4321 9990-10060 Sentence denotes The HIV-1Lai/Bal-Env chimeric virus uses CCR5 as a secondary receptor.
T61 9990-10060 Sentence denotes The HIV-1Lai/Bal-Env chimeric virus uses CCR5 as a secondary receptor.
T4322 10061-10210 Sentence denotes The infectious molecular clone of HIV-198IN22 was constructed using DNA extracted from PBMC that were infected with a primary isolate of HIV-198IN22.
T62 10061-10210 Sentence denotes The infectious molecular clone of HIV-198IN22 was constructed using DNA extracted from PBMC that were infected with a primary isolate of HIV-198IN22.
T4323 10211-10352 Sentence denotes HIV-1Lai/Bal-Env and HIV-198IN22 mutant viruses were constructed by introducing point mutations using standard PCR-based mutagenesis methods.
T63 10211-10352 Sentence denotes HIV-1Lai/Bal-Env and HIV-198IN22 mutant viruses were constructed by introducing point mutations using standard PCR-based mutagenesis methods.
T4646 10354-10379 Sentence denotes siRNA transfection of MDM
T64 10354-10379 Sentence denotes siRNA transfection of MDM
T4647 10380-10487 Sentence denotes An siRNA was constructed (Ambion Inc., www.ambion.com) to target a sequence unique to the NFAT5 transcript:
T65 10380-10487 Sentence denotes An siRNA was constructed (Ambion Inc., www.ambion.com) to target a sequence unique to the NFAT5 transcript:
T4648 10488-10535 Sentence denotes 5′-CAACATGCCTGGAATTCAA-3′ (nt 335 to 353) [31].
T66 10488-10535 Sentence denotes 5′-CAACATGCCTGGAATTCAA-3′ (nt 335 to 353) [31].
T4649 10536-10685 Sentence denotes As described, a control for non-specific siRNA effects, we used an siRNA targeting the green fluorescent protein (GFP), 5′- GGCTACGTCCAGGAGCGCACC-3′.
T67 10536-10685 Sentence denotes As described, a control for non-specific siRNA effects, we used an siRNA targeting the green fluorescent protein (GFP), 5′- GGCTACGTCCAGGAGCGCACC-3′.
T4650 10686-10964 Sentence denotes MDM were transfected in 6-well plates using 1 µM of the indicated siRNA in siPORT NeoFX transfection reagent (Ambion Inc., www.ambion.com), prepared as recommended by the manufacturer, in a final volume of 750 µl in Macrophage-SFM medium plus 5% heat-inactivated human AB serum.
T68 10686-10964 Sentence denotes MDM were transfected in 6-well plates using 1 µM of the indicated siRNA in siPORT NeoFX transfection reagent (Ambion Inc., www.ambion.com), prepared as recommended by the manufacturer, in a final volume of 750 µl in Macrophage-SFM medium plus 5% heat-inactivated human AB serum.
T4651 10965-11066 Sentence denotes The cultures were left at 37°C overnight after which cells were washed and incubated in fresh medium.
T69 10965-11066 Sentence denotes The cultures were left at 37°C overnight after which cells were washed and incubated in fresh medium.
T4652 11067-11192 Sentence denotes MDM were transfected two times for efficient knock down of NFAT5 expression before infection experiments were performed [31].
T70 11067-11192 Sentence denotes MDM were transfected two times for efficient knock down of NFAT5 expression before infection experiments were performed [31].
T5068 11194-11229 Sentence denotes Stable THP-1 cells expressing shRNA
T71 11194-11229 Sentence denotes Stable THP-1 cells expressing shRNA
T5069 11230-11915 Sentence denotes The lentiviral plasmid pLKO.1 expressing shRNA targeting human MyD88 was purchased from Open Biosystems (www.openbiosystems.com) and was validated in our laboratory. shRNA targeting human IRAK1 (forward primer 5′-CCGGAGCAGCTGTCCAGGTTTCGTCTCATAAAACCTGGACAGCTGCTCCTTTTTG-3′, reverse primer 5′-AATTCAAAAAGGAGCAGCTGTCCAGGTTTTATGAGACGAAACCTGGACAGCTGCT-3′ mRNA (IRAK1 mRNA target sequence is underlined) and human TRAF6 (forward primer 5′-CCGGAGAAACCTGTTGTGATTCGTCTCATAAATCACAACAGGTTTCTCCTTTTTG-3′, reverse primer 5′-AATTCAAAAAGGAGAAACCTGTTGTGATTTATGAGACGAATCACAACAGGTTTCT-3′ (TRAF6 mRNA target sequence is underlined) were designed in our laboratory and were cloned into the pLKO.1 plasmid.
T72 11230-11915 Sentence denotes The lentiviral plasmid pLKO.1 expressing shRNA targeting human MyD88 was purchased from Open Biosystems (www.openbiosystems.com) and was validated in our laboratory. shRNA targeting human IRAK1 (forward primer 5′-CCGGAGCAGCTGTCCAGGTTTCGTCTCATAAAACCTGGACAGCTGCTCCTTTTTG-3′, reverse primer 5′-AATTCAAAAAGGAGCAGCTGTCCAGGTTTTATGAGACGAAACCTGGACAGCTGCT-3′ mRNA (IRAK1 mRNA target sequence is underlined) and human TRAF6 (forward primer 5′-CCGGAGAAACCTGTTGTGATTCGTCTCATAAATCACAACAGGTTTCTCCTTTTTG-3′, reverse primer 5′-AATTCAAAAAGGAGAAACCTGTTGTGATTTATGAGACGAATCACAACAGGTTTCT-3′ (TRAF6 mRNA target sequence is underlined) were designed in our laboratory and were cloned into the pLKO.1 plasmid.
T5070 11916-12201 Sentence denotes Lentiviruses encoding shRNA sequences were generated by transfecting the packaging cell line HEK-293T with the shRNA-encoding pLKO.1 plasmids in combination with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G using Effectene transfection reagent (Qiagen, www.qiagen.com).
T73 11916-12201 Sentence denotes Lentiviruses encoding shRNA sequences were generated by transfecting the packaging cell line HEK-293T with the shRNA-encoding pLKO.1 plasmids in combination with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G using Effectene transfection reagent (Qiagen, www.qiagen.com).
T74 12202-12307 Sentence denotes Supernatants were collected 48 hours post-transfection, clarified by centrifugation, and stored at −80°C.
T5071 12202-12550 Sentence denotes Supernatants were collected 48 hours post-transfection, clarified by centrifugation, and stored at −80°C. THP-1 cells were transduced with the lentiviral particles by culturing the cells with supernatants from the virus-producing cells in the presence of 8 µg/ml polybrene (Millipore, www.millipore.com) and spinoculation for two hours at 2000 RPM.
T75 12308-12550 Sentence denotes THP-1 cells were transduced with the lentiviral particles by culturing the cells with supernatants from the virus-producing cells in the presence of 8 µg/ml polybrene (Millipore, www.millipore.com) and spinoculation for two hours at 2000 RPM.
T5072 12551-12646 Sentence denotes Successfully transduced cells were selected and expanded by treatment with 0.8 µg/ml puromycin.
T76 12551-12646 Sentence denotes Successfully transduced cells were selected and expanded by treatment with 0.8 µg/ml puromycin.
T5600 12648-12659 Sentence denotes MTb culture
T77 12648-12659 Sentence denotes MTb culture
T5601 12660-12913 Sentence denotes The MTb clinical strain CDC1551 was prepared by adding 100 µl of frozen bacteria stock into 10 ml of Middlebrook 7H9 medium (Difco BD, www.bd.com) supplemented with albumin dextrose complex (ADC) and 0.05% Tween 80 (Sigma-Aldrich, www.sigmaaldrich.com).
T78 12660-12913 Sentence denotes The MTb clinical strain CDC1551 was prepared by adding 100 µl of frozen bacteria stock into 10 ml of Middlebrook 7H9 medium (Difco BD, www.bd.com) supplemented with albumin dextrose complex (ADC) and 0.05% Tween 80 (Sigma-Aldrich, www.sigmaaldrich.com).
T5602 12914-13022 Sentence denotes The cultures were grown to an OD650 of 0.4 at 37°C to ensure that they were in the logarithmic growth phase.
T79 12914-13022 Sentence denotes The cultures were grown to an OD650 of 0.4 at 37°C to ensure that they were in the logarithmic growth phase.
T5603 13023-13181 Sentence denotes Bacteria were then plated, washed with PBS, resuspended in PBS, and passed through a 5 µm filter to ensure that the bacteria were in a single cell suspension.
T80 13023-13181 Sentence denotes Bacteria were then plated, washed with PBS, resuspended in PBS, and passed through a 5 µm filter to ensure that the bacteria were in a single cell suspension.
T5604 13182-13378 Sentence denotes Bacterial cell numbers were determined by measurement of OD650 before further dilution with RPMI 1640 medium for cell infection studies at 10∶1 PBMC∶ bacilli or 1∶1 MDM∶ bacilli and THP-1∶bacilli.
T81 13182-13378 Sentence denotes Bacterial cell numbers were determined by measurement of OD650 before further dilution with RPMI 1640 medium for cell infection studies at 10∶1 PBMC∶ bacilli or 1∶1 MDM∶ bacilli and THP-1∶bacilli.
T5605 13379-13597 Sentence denotes Colony-forming unit (CFU) analysis was performed and on days 4 and 7 the average CFU counts were 6×103 and 5×104, respectively, confirming that mycobacteria levels increased over the course of infection of primary MDM.
T82 13379-13597 Sentence denotes Colony-forming unit (CFU) analysis was performed and on days 4 and 7 the average CFU counts were 6×103 and 5×104, respectively, confirming that mycobacteria levels increased over the course of infection of primary MDM.
T5993 13599-13611 Sentence denotes Western blot
T83 13599-13611 Sentence denotes Western blot
T5994 13612-13829 Sentence denotes Whole cell extracts were collected with lysis buffer containing 150 mM NaCl, 50 mM Tris–HCl, pH 7.5, 1% Triton, 10% glycerol, and 1 tablet of Complete EDTA-free Protease Inhibitor Cocktail (Roche) per 25 ml of buffer.
T84 13612-13829 Sentence denotes Whole cell extracts were collected with lysis buffer containing 150 mM NaCl, 50 mM Tris–HCl, pH 7.5, 1% Triton, 10% glycerol, and 1 tablet of Complete EDTA-free Protease Inhibitor Cocktail (Roche) per 25 ml of buffer.
T5995 13830-13959 Sentence denotes Extracts were boiled for 5 min in 1× Laemmli sample buffer with 5% v/v 2-mercaptoethanol and proteins were separated by SDS-PAGE.
T85 13830-13959 Sentence denotes Extracts were boiled for 5 min in 1× Laemmli sample buffer with 5% v/v 2-mercaptoethanol and proteins were separated by SDS-PAGE.
T5996 13960-14042 Sentence denotes The gel was transferred to a nitrocellulose Trans-Blot Transfer Membrane (BioRad).
T86 13960-14042 Sentence denotes The gel was transferred to a nitrocellulose Trans-Blot Transfer Membrane (BioRad).
T5997 14043-14215 Sentence denotes The blot was then blocked for 1 h at 37°C in a solution of 4% BSA (Sigma) and 0.1% Tween-20 (BioRad) in a buffer containing 50 mM Tris and 150 mM NaCl at pH 7.6 (BSA/TBST).
T87 14043-14215 Sentence denotes The blot was then blocked for 1 h at 37°C in a solution of 4% BSA (Sigma) and 0.1% Tween-20 (BioRad) in a buffer containing 50 mM Tris and 150 mM NaCl at pH 7.6 (BSA/TBST).
T5998 14216-14465 Sentence denotes Primary incubation was carried out with a1∶200 dilution of rabbit anti-NFAT5 antibody (H-300) (Santa Cruz Biotechnology) and a 1∶500 dilution of goat anti-Lamin-B1 antibody (sc-6217; Santa Cruz Biotechnology) in BSA/TBST for 2 h at room temperature.
T88 14216-14465 Sentence denotes Primary incubation was carried out with a1∶200 dilution of rabbit anti-NFAT5 antibody (H-300) (Santa Cruz Biotechnology) and a 1∶500 dilution of goat anti-Lamin-B1 antibody (sc-6217; Santa Cruz Biotechnology) in BSA/TBST for 2 h at room temperature.
T5999 14466-14630 Sentence denotes The blot was washed 3×5 min in TBST and incubated in 1∶6000 donkey anti-goat-HRP (Santa Cruz Biotechnology) or goat anti-rabbit-HRP (BioRad) as appropriate for 1 h.
T89 14466-14630 Sentence denotes The blot was washed 3×5 min in TBST and incubated in 1∶6000 donkey anti-goat-HRP (Santa Cruz Biotechnology) or goat anti-rabbit-HRP (BioRad) as appropriate for 1 h.
T6000 14631-14748 Sentence denotes The blot was again washed 3×5 min in TBST and developed with SuperSignal West Pico Chemiluminescent Reagent (Pierce).
T90 14631-14748 Sentence denotes The blot was again washed 3×5 min in TBST and developed with SuperSignal West Pico Chemiluminescent Reagent (Pierce).
T6529 14750-14766 Sentence denotes Quantitative PCR
T91 14750-14766 Sentence denotes Quantitative PCR
T6530 14767-14892 Sentence denotes The mRNA expression levels were determined by SYBR Green-based real-time PCR (Applied Biosystems, www.appliedbiosystems.com).
T92 14767-14892 Sentence denotes The mRNA expression levels were determined by SYBR Green-based real-time PCR (Applied Biosystems, www.appliedbiosystems.com).
T6531 14893-14998 Sentence denotes The reaction conditions were 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min.
T93 14893-14998 Sentence denotes The reaction conditions were 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min.
T6532 14999-15102 Sentence denotes The results were normalized using β-actin mRNA as an internal control and expressed as relative values.
T94 14999-15102 Sentence denotes The results were normalized using β-actin mRNA as an internal control and expressed as relative values.
T6677 15104-15124 Sentence denotes Statistical analysis
T95 15104-15124 Sentence denotes Statistical analysis
T6678 15125-15179 Sentence denotes Where applicable, results are expressed as mean ± SEM.
T96 15125-15179 Sentence denotes Where applicable, results are expressed as mean ± SEM.
T6679 15180-15332 Sentence denotes Comparison between two groups was performed using the paired Student t-Test with the aid of Microsoft Excel software. p≤0.05 was considered significant.
T97 15180-15332 Sentence denotes Comparison between two groups was performed using the paired Student t-Test with the aid of Microsoft Excel software. p≤0.05 was considered significant.
T98 15334-15341 Sentence denotes Results
T6766 15343-15405 Sentence denotes MTb increases HIV-1 LTR activity of HIV-1 subtypes B, C, and E
T99 15343-15405 Sentence denotes MTb increases HIV-1 LTR activity of HIV-1 subtypes B, C, and E
T6767 15406-15685 Sentence denotes To compare the functional impact of MTb stimulation on subtype-specific HIV-1 LTR activity, we first constructed reporter plasmids containing viral subtype B, C, and E LTRs (−208 to + 64 nt relative to the transcription start site) linked to the firefly luciferase reporter gene.
T100 15406-15685 Sentence denotes To compare the functional impact of MTb stimulation on subtype-specific HIV-1 LTR activity, we first constructed reporter plasmids containing viral subtype B, C, and E LTRs (−208 to + 64 nt relative to the transcription start site) linked to the firefly luciferase reporter gene.
T6768 15686-15833 Sentence denotes After transfection of the monocytic THP-1 cell line with these plasmids, cells were stimulated with an irradiated whole cell lysate of MTb (H37Rv).
T101 15686-15833 Sentence denotes After transfection of the monocytic THP-1 cell line with these plasmids, cells were stimulated with an irradiated whole cell lysate of MTb (H37Rv).
T6769 15834-15983 Sentence denotes We note that MTb lysate induces inflammatory responses in monocytes that resemble those induced in response to live MTb (see for example, [35]–[37]).
T102 15834-15983 Sentence denotes We note that MTb lysate induces inflammatory responses in monocytes that resemble those induced in response to live MTb (see for example, [35]–[37]).
T6770 15984-16167 Sentence denotes Upon stimulation, the B, C, and E LTR-driven reporters demonstrated a significant enhancement in luciferase activity (Figure 1A) and the magnitude of this effect was subtype-specific.
T103 15984-16167 Sentence denotes Upon stimulation, the B, C, and E LTR-driven reporters demonstrated a significant enhancement in luciferase activity (Figure 1A) and the magnitude of this effect was subtype-specific.
T6771 16168-16426 Sentence denotes Subtype C LTRs displayed the strongest activity, while the LTRs from subtype E isolates consistently showed the weakest activity (Figure 1A), consistent with previous studies demonstrating subtype-specific LTR activity that used TNF as a stimulus [38], [39].
T104 16168-16426 Sentence denotes Subtype C LTRs displayed the strongest activity, while the LTRs from subtype E isolates consistently showed the weakest activity (Figure 1A), consistent with previous studies demonstrating subtype-specific LTR activity that used TNF as a stimulus [38], [39].
T105 16427-16551 Sentence denotes 10.1371/journal.ppat.1002620.g001 Figure 1 NFAT5 interaction with the LTR is important for MTb-induced HIV-1 transcription.
T21040 16471-16551 Sentence denotes NFAT5 interaction with the LTR is important for MTb-induced HIV-1 transcription.
T106 16552-16639 Sentence denotes (A) MTb stimulation increases activity of LTRs derived from HIV-1 subtypes B, C, and E.
T21041 16552-16794 Sentence denotes (A) MTb stimulation increases activity of LTRs derived from HIV-1 subtypes B, C, and E. HIV-1 LTRs (−208 to +64 nt relative to the transcription start site) from representative subtype B, C, and E viral isolates were cloned into plasmid pGL3.
T107 16640-16794 Sentence denotes HIV-1 LTRs (−208 to +64 nt relative to the transcription start site) from representative subtype B, C, and E viral isolates were cloned into plasmid pGL3.
T21042 16795-16975 Sentence denotes THP-1 cells (0.8×106/ml) were transfected with each reporter plasmid (0.3 µg/ml) plus the Renilla luciferase control plasmid pRL-TK (0.03 µg/ml) and incubated at 37°C for 16 hours.
T108 16795-16975 Sentence denotes THP-1 cells (0.8×106/ml) were transfected with each reporter plasmid (0.3 µg/ml) plus the Renilla luciferase control plasmid pRL-TK (0.03 µg/ml) and incubated at 37°C for 16 hours.
T21043 16976-17097 Sentence denotes Cells were then either left untreated or treated with 10 µg/ml MTb lysate for 8 hours before termination of the cultures.
T109 16976-17097 Sentence denotes Cells were then either left untreated or treated with 10 µg/ml MTb lysate for 8 hours before termination of the cultures.
T21044 17098-17181 Sentence denotes In the histogram, open bars represent individual LTR activities in untreated cells.
T110 17098-17181 Sentence denotes In the histogram, open bars represent individual LTR activities in untreated cells.
T21045 17182-17275 Sentence denotes Light grey bars represent mean values of LTR activities from each subtype in untreated cells.
T111 17182-17275 Sentence denotes Light grey bars represent mean values of LTR activities from each subtype in untreated cells.
T21046 17276-17462 Sentence denotes Black bars represent individual LTR activities in MTb lysate-treated cells, and dark grey bars represent mean values of LTR activities from each subtype in cells treated with MTb lysate.
T112 17276-17462 Sentence denotes Black bars represent individual LTR activities in MTb lysate-treated cells, and dark grey bars represent mean values of LTR activities from each subtype in cells treated with MTb lysate.
T21047 17463-17634 Sentence denotes LTR transcriptional activity for all of the representative LTRs tested was significantly increased in cultures treated with MTb lysate in comparison to untreated cultures.
T113 17463-17634 Sentence denotes LTR transcriptional activity for all of the representative LTRs tested was significantly increased in cultures treated with MTb lysate in comparison to untreated cultures.
T21048 17635-17921 Sentence denotes Results are from three independent experiments performed in duplicate (*, p<0.05; **, p<0.01 as compared to unstimulated cultures). (B) Specific disruption of the NFAT5 binding site significantly reduces LTR-reporter gene activity in monocytic cells in response to MTb lysate treatment.
T114 17635-17921 Sentence denotes Results are from three independent experiments performed in duplicate (*, p<0.05; **, p<0.01 as compared to unstimulated cultures). (B) Specific disruption of the NFAT5 binding site significantly reduces LTR-reporter gene activity in monocytic cells in response to MTb lysate treatment.
T21049 17922-18121 Sentence denotes THP-1 cells were transfected with luciferase expression vectors encoding nucleotides 208 to +64 of the wild-type HIV-1Lai LTR and an HIV-1Lai LTR containing the NFAT5 binding site mutations (N5-Mut).
T115 17922-18121 Sentence denotes THP-1 cells were transfected with luciferase expression vectors encoding nucleotides 208 to +64 of the wild-type HIV-1Lai LTR and an HIV-1Lai LTR containing the NFAT5 binding site mutations (N5-Mut).
T116 18122-18222 Sentence denotes After 16 hours, the cells were left untreated or exposed to 10 µg/ml MTb lysate for 8 hours at 37°C.
T21050 18122-18419 Sentence denotes After 16 hours, the cells were left untreated or exposed to 10 µg/ml MTb lysate for 8 hours at 37°C. Disruption of NFAT5 binding to the enhancer region significantly suppressed LTR-driven reporter gene expression in comparison to the wild-type LTR when cells were treated with MTb lysate (p<0.01).
T117 18223-18419 Sentence denotes Disruption of NFAT5 binding to the enhancer region significantly suppressed LTR-driven reporter gene expression in comparison to the wild-type LTR when cells were treated with MTb lysate (p<0.01).
T21051 18420-18508 Sentence denotes LTR activity was also suppressed in the untreated cells but to a lesser extent (p<0.05).
T118 18420-18508 Sentence denotes LTR activity was also suppressed in the untreated cells but to a lesser extent (p<0.05).
T21052 18509-18657 Sentence denotes Results are from three independent experiments performed in duplicate and adjusted to Renilla luciferase control expression (*, p<0.05; **, p<0.01).
T119 18509-18657 Sentence denotes Results are from three independent experiments performed in duplicate and adjusted to Renilla luciferase control expression (*, p<0.05; **, p<0.01).
T21053 18658-18855 Sentence denotes Nucleotide sequences representing the wild-type and NFAT5 binding site-mutated HIV-1Lai LTRs are shown at the bottom of the figure. (C) MTb lysate increases NFAT5 protein levels in monocytic cells.
T120 18658-18855 Sentence denotes Nucleotide sequences representing the wild-type and NFAT5 binding site-mutated HIV-1Lai LTRs are shown at the bottom of the figure. (C) MTb lysate increases NFAT5 protein levels in monocytic cells.
T121 18856-18958 Sentence denotes THP-1 cells were left untreated (control) or exposed to 10 µg/ml MTb lysate for 8 or 24 hours at 37°C.
T21054 18856-19048 Sentence denotes THP-1 cells were left untreated (control) or exposed to 10 µg/ml MTb lysate for 8 or 24 hours at 37°C. Whole cell extracts were collected and analyzed by western blot with anti-NFAT5 antibody.
T122 18959-19048 Sentence denotes Whole cell extracts were collected and analyzed by western blot with anti-NFAT5 antibody.
T21055 19049-19117 Sentence denotes An antibody directed against Lamin-B1 was used as a loading control.
T123 19049-19117 Sentence denotes An antibody directed against Lamin-B1 was used as a loading control.
T21056 19118-19325 Sentence denotes The histogram shows densitometric analysis of the NFAT5 bands from the western blot autoradiograph displayed and values represent mean band intensities at 0, 8, and 24 hours post-stimulation with MTb lysate.
T124 19118-19325 Sentence denotes The histogram shows densitometric analysis of the NFAT5 bands from the western blot autoradiograph displayed and values represent mean band intensities at 0, 8, and 24 hours post-stimulation with MTb lysate.
T7299 19327-19442 Sentence denotes Specific disruption of NFAT5 binding impairs LTR-driven transcription in THP-1 cells in response to MTb stimulation
T125 19327-19442 Sentence denotes Specific disruption of NFAT5 binding impairs LTR-driven transcription in THP-1 cells in response to MTb stimulation
T7300 19443-19779 Sentence denotes Although the transcription factor NFAT5 binds to a core NF-κB binding motif in the HIV-1 LTR enhancer region of subtype B, when two thymines (TT) are changed to cytosines (CC) in the proximal NF-κB binding motif (named N5-Mut) (bottom of Figure 1B), the binding of NFAT5 can be disrupted while leaving binding of NF-κB unperturbed [31].
T126 19443-19779 Sentence denotes Although the transcription factor NFAT5 binds to a core NF-κB binding motif in the HIV-1 LTR enhancer region of subtype B, when two thymines (TT) are changed to cytosines (CC) in the proximal NF-κB binding motif (named N5-Mut) (bottom of Figure 1B), the binding of NFAT5 can be disrupted while leaving binding of NF-κB unperturbed [31].
T7301 19780-19975 Sentence denotes We examined the requirement of NFAT5 for MTb-induced LTR activity by transfecting a wild-type and NFAT5 mutant LTR reporter construct into THP-1 cells, followed by stimulation with an MTb lysate.
T127 19780-19975 Sentence denotes We examined the requirement of NFAT5 for MTb-induced LTR activity by transfecting a wild-type and NFAT5 mutant LTR reporter construct into THP-1 cells, followed by stimulation with an MTb lysate.
T7302 19976-20160 Sentence denotes As shown in Figure 1B, in the absence of MTb lysate stimulation the activity of the NFAT5 binding site-mutant LTR was significantly reduced (p<0.05) in comparison to the wild-type LTR.
T128 19976-20160 Sentence denotes As shown in Figure 1B, in the absence of MTb lysate stimulation the activity of the NFAT5 binding site-mutant LTR was significantly reduced (p<0.05) in comparison to the wild-type LTR.
T7303 20161-20329 Sentence denotes When the NFAT5 binding site-mutant LTR was examined in THP-1 cells stimulated with the MTb lysate, its activity was reduced to an even more significant extent (p<0.01).
T129 20161-20329 Sentence denotes When the NFAT5 binding site-mutant LTR was examined in THP-1 cells stimulated with the MTb lysate, its activity was reduced to an even more significant extent (p<0.01).
T7304 20330-20539 Sentence denotes To determine whether MTb lysate stimulation directly enhances NFAT5 expression in THP-1 cells, we stimulated cells for 8 or 24 hours or left them unstimulated and examined NFAT5 protein levels by western blot.
T130 20330-20539 Sentence denotes To determine whether MTb lysate stimulation directly enhances NFAT5 expression in THP-1 cells, we stimulated cells for 8 or 24 hours or left them unstimulated and examined NFAT5 protein levels by western blot.
T7305 20540-20808 Sentence denotes As shown in Figure 1C, we found that NFAT5 protein levels steadily increased in response to MTb lysate stimulation, revealing that TLR engagement by MTb results in enhanced levels of NFAT5, consistent with its playing a role in MTb-induced activation of the HIV-1 LTR.
T131 20540-20808 Sentence denotes As shown in Figure 1C, we found that NFAT5 protein levels steadily increased in response to MTb lysate stimulation, revealing that TLR engagement by MTb results in enhanced levels of NFAT5, consistent with its playing a role in MTb-induced activation of the HIV-1 LTR.
T8129 20810-20854 Sentence denotes MTb increases NFAT5 mRNA levels in human MDM
T132 20810-20854 Sentence denotes MTb increases NFAT5 mRNA levels in human MDM
T8130 20855-21096 Sentence denotes Given that macrophages, which are the primary target of MTb infection, are also a major reservoir of HIV-1 as infection progresses [40], we next investigated whether MTb is able to directly enhance NFAT5 mRNA expression in primary human MDM.
T133 20855-21096 Sentence denotes Given that macrophages, which are the primary target of MTb infection, are also a major reservoir of HIV-1 as infection progresses [40], we next investigated whether MTb is able to directly enhance NFAT5 mRNA expression in primary human MDM.
T8131 21097-21297 Sentence denotes We prepared MDM from five normal donors, stimulated the cells with the MTb lysate or left them unstimulated, and measured NFAT5 gene expression levels by quantitative real-time PCR at 24 and 48 hours.
T134 21097-21297 Sentence denotes We prepared MDM from five normal donors, stimulated the cells with the MTb lysate or left them unstimulated, and measured NFAT5 gene expression levels by quantitative real-time PCR at 24 and 48 hours.
T8132 21298-21521 Sentence denotes We also investigated whether HIV-1 infection is capable of inducing NFAT5 mRNA synthesis by infecting MDM with live or heat-inactivated R5-tropic representatives of subtype B (HIV-1Bal), C (HIV-198IN22), or E (HIV-192TH64).
T135 21298-21521 Sentence denotes We also investigated whether HIV-1 infection is capable of inducing NFAT5 mRNA synthesis by infecting MDM with live or heat-inactivated R5-tropic representatives of subtype B (HIV-1Bal), C (HIV-198IN22), or E (HIV-192TH64).
T8133 21522-21765 Sentence denotes As shown in Figure 2A, stimulation with MTb lysate significantly increased NFAT5 mRNA levels at 24 (p<0.05) and 48 (p<0.01) hours, whereas infection with viable or heat-inactivated HIV-1 isolates did not increase NFAT5 mRNA levels (Figure 2A).
T136 21522-21765 Sentence denotes As shown in Figure 2A, stimulation with MTb lysate significantly increased NFAT5 mRNA levels at 24 (p<0.05) and 48 (p<0.01) hours, whereas infection with viable or heat-inactivated HIV-1 isolates did not increase NFAT5 mRNA levels (Figure 2A).
T8134 21766-21925 Sentence denotes Thus, MTb specifically enhances NFAT5 mRNA expression in human MDM, and this response continues to increase for at least 48 hours post-stimulation (Figure 2A).
T137 21766-21925 Sentence denotes Thus, MTb specifically enhances NFAT5 mRNA expression in human MDM, and this response continues to increase for at least 48 hours post-stimulation (Figure 2A).
T138 21926-22077 Sentence denotes 10.1371/journal.ppat.1002620.g002 Figure 2 MTb induces NFAT5 gene expression and knockdown of NFAT5 impairs HIV-1 replication during MTb co-infection.
T22397 21970-22077 Sentence denotes MTb induces NFAT5 gene expression and knockdown of NFAT5 impairs HIV-1 replication during MTb co-infection.
T14766 21970-41008 Sentence denotes MTb induces NFAT5 gene expression and knockdown of NFAT5 impairs HIV-1 replication during MTb co-infection. (A) MTb but not HIV-1 increases NFAT5 mRNA synthesis in human macrophages. MDM were isolated from PBMC obtained from four normal donors. The cells were incubated for 24 or 48 hours with heat-inactivated or live R5-tropic HIV-1 subtype B, C, or E isolates, live MTb strain CDC1551 (1∶1 MDM∶bacilli), or left untreated. Quantitative real-time PCR analysis of NFAT5 mRNA expression levels revealed that the low, constitutively present level of NFAT5 mRNA expressed in untreated cells was not affected by exposure to live or inactivated HIV-1, but was significantly increased at both 24 (*, p<0.05) and 48 (**, p<0.01) hours following infection with MTb. (B) NFAT5-specific siRNA effectively suppresses NFAT5 expression in MDM in both the absence and presence of MTb co-infection. MDM (1×106 cells/well in a 6-well plate) from four normal donors were transfected with siRNA specific for NFAT5 or, as a control, GFP, and were then infected with MTb (1∶1 MDM∶bacilli) or left uninfected. In the cells transfected with control siRNA, MTb infection significantly increased NFAT5 expression in comparison to uninfected cells (open bars). When MDM were transfected with NFAT5-specific siRNA, both constitutively expressed (p = 0.048) and MTb-induced (p = 0.021) NFAT5 mRNA were significantly suppressed (grey bars). (C) HIV-1 subtype B replication is significantly suppressed in the presence of MTb co-infection when NFAT5 expression is abrogated in human MDM. MDM from four normal donors were transfected with NFAT5-specific and control siRNAs as described in 2B. Cells were then infected with HIV-1Lai/Bal-env followed by co-infection with MTb CDC1551. Virus replication was measured at days 6, 9, and 12 post-infection. At day 12, virus replication was significantly reduced in cells transfected with NFAT5-specific siRNA in comparison to cells transfected with control siRNA (*, p<0.05). We monitored the morphology of cells within the infected cultures and did not observe noticeable macrophage necrosis even at the final timepoint of viral harvest. RNAi directed against NFAT5 inhibits replication of subtype B HIV-1 To extend the results we obtained in the reporter assays to a physiological TB/HIV co-infection model, we next tested the effect of siRNA-mediated ablation of NFAT5 mRNA levels in MDM co-infected with a subtype B HIV-1Lai infectious molecular clone and a clinical isolate of MTb. To perform this experiment, we first constructed an HIV-1Lai clone bearing the CCR5-tropic envelope region of HIV-1Bal (HIV-1Lai/Bal-env) so that it would efficiently infect primary MDM. We used this approach to ensure that our analysis of the roles of NF-κB and NFAT5 in MTb-induced HIV-1 replication could be interpreted in the proper context of previous research findings that examined LTR regulation in the context of full-length viral replication [41]–[46]. The HIV-1Lai/Bal-Env infectious clone is isogenic for the entire sequence of the parental HIV-1Lai infectious clone except for the substitution of the HIV-1Bal envelope co-receptor binding region in place of the HIV-1Lai envelope co-receptor binding region. We note that we confirmed that HIV-1Lai/Bal-Env grew in PBMC at a similar rate to wild-type HIV-1Bal, indicating proper co-receptor engagement and internalization of this infectious clone (data not shown). Next, we knocked down NFAT5 mRNA levels in MDM using an siRNA that suppresses both NFAT5 mRNA and NFAT5 protein levels [31]. As shown in Figure 2, transfection of the siRNA specific for NFAT5 reduces NFAT5 mRNA levels in both MTb-uninfected (p = 0.048) and MTb-infected (p = 0.021) MDM as compared to transfection of control GFP siRNA into MTb-uninfected or -infected MDM (Figure 2B). We note that although siRNA normally is effective for 48–72 hours in cell lines that divide rapidly, in human MDM, which are non-dividing cells, siRNA to host factors remains detectable and functional up to at least 15 days post-transfection [47]. MDM in which NFAT5 expression had been inhibited with NFAT5 siRNA or that were transfected with control GFP siRNA were then infected with 1000 TCID50 of HIV-1Lai/Bal-env. After overnight virus infection, the cells were then co-infected with the MTb clinical strain CDC1551. Free virus levels were then measured in culture supernatants from MDM transfected with NFAT5-specific siRNA or GFP control siRNA at days 6, 9 and 12 post-HIV-1 infection to measure the impact of NFAT5 inhibition on MTb-induced HIV replication. As shown in Figure 2C, HIV-1 replication was suppressed at days 6 and 9 post-infection in the NFAT5 siRNA-treated cells as compared to cells treated with control siRNA, and it was significantly inhibited by day 12 post-infection (p<0.05) (Figure 2C). Thus, knock down of NFAT5 expression significantly impairs HIV-1 subtype B replication in MDM co-infected with MTb. NF-κB and NFAT5 binding to the HIV-1 LTR can be specifically disrupted To demonstrate that the impact of NFAT5 silencing on MTb-induced viral replication was a direct effect due to modulation of recruitment of NFAT5 to the HIV-1 LTR and not due to secondary, NFAT5-regulated effects, we set out to disrupt NFAT5 binding to the viral LTR in the context of HIV-1/MTb co-infection. This series of experiments also allowed us to dissect the relative importance of NFAT5 and NF-κB binding to the HIV-1 LTR in MTb regulation of HIV-1 replication. To perform these experiments, we constructed a panel of infectious HIV-1Lai/Bal-env molecular clones where either the NFAT5 site was specifically disrupted or the two NF-κB binding sites were either individually or dually disrupted (Figure 3A). Specifically, we constructed a clone in which the NFAT5 binding site was mutated (named HIV-1Lai/Bal-env-N5-Mut) by changing the CC dinucleotide to TT in the core NFAT5 binding site and introduced substitution mutations into the NF-κB/NFAT5 shared binding element that we predicted would disrupt NF-κB binding but preserve NFAT5 binding. Specifically, we mutated two guanines (GG) in the κB I site and changed them to thymine and adenine (TA) (HIV-1Lai/Bal-env-κB I-mut) and also introduced the same GG to TA change in the second, more distal NF-κB site (κB II) in HIV-1Lai/Bal-env (HIV-1Lai/Bal-env-κB II-Mut). We also created a double NF-κB site mutant virus (HIV-1Lai/Bal-env-κB I+II-Mut) in order to test the impact of complete disruption of NF-κB binding to the HIV-1 LTR on MTb regulation. 10.1371/journal.ppat.1002620.g003 Figure 3 Specific disruption of NFAT5 or NF-κB binding sites in the LTR of HIV-1 subtype B. (A) Mutations introduced into a HIV-1Lai/Bal-env infectious molecular clone. NF-κB and NFAT5 binding site mutations were introduced into the LTR of the HIV-1Lai/Bal-env-wild type (WT) infectious molecular clone. The NF-κB binding site mutants (HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut), and NFAT5 binding site mutant (HIV-1Lai/Bal-env-N5-Mut) are shown along with the HIV-1Lai/Bal-env-WT sequence. Mutations were introduced into the 3′ LTR of the HIV-1Lai/Bal-env–WT proviral sequence. (B) Specific mutation of the NFAT5 binding site abolishes NFAT5 binding to the viral LTR but does not affect NF-κB p50/p65 binding to the overlapping NF-κB binding site. Quantitative DNaseI footprinting analysis is shown using HIV-1 LTR fragments (-262 to +4 nt relative to the transcription start site) from HIV-1Lai/Bal-env-WT and HIV-1Lai/Bal-env-N5-Mut and increasing concentrations of recombinant NF-κB (p50/p65) (25 ng, 100 ng, and 500 ng), or NFAT5 (10 ng, 50 ng, and 250 ng). The regions that are protected from DNase I cleavage by the binding of NF-κB and NFAT5 are indicated with a bars. (C) Specific disruption of the HIV-1 subtype B NF-κB binding sites effectively abrogates recombinant p50/p65 binding. Quantitative DNaseI footprinting analysis is shown of nucleotides −262 to +4 from HIV-1Lai/Bal-env-WT, HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, and HIV-1Lai/Bal-env-κB I+II-Mut and increasing concentrations of recombinant NF-κB (p50/p65) (25 ng, 100 ng, and 500 ng). The regions that are protected from DNase I cleavage by the binding of recombinant NF-κB are indicated with a bars. (D) Specific disruption of the HIV-1 subtype B NF-κB binding site does not inhibit but enhances NFAT5 binding to this region. Quantitative DNaseI footprinting analysis is shown of nucleotides −262 to +4 from HIV-1Lai/Bal-env-WT, HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, and HIV-1Lai/Bal-env-κB I+II-Mut and increasing concentrations of recombinant NFAT5 (10 ng, 50 ng, and 250 ng). The regions that are protected from DNase I cleavage by the binding of recombinant NFAT5 are indicated with a bars. To determine the specificity of these substitutions upon NFAT5 and NF-κB p50/p65 binding to the LTR, we performed a quantitative DNase I footprinting analysis using the wild-type LTR or mutant LTRs in combination with increasing concentrations of recombinant NFAT5 or NF-κB p50/p65 proteins. As shown in Figure 3B, the N5-Mut LTR could not bind NFAT5 (compare lanes 12–15 and lanes 17–20), but p50/p65 binding was not impaired (compare lanes 2–5 with lanes 7–10). By contrast, changing the GG dinucleotide to TA in either NF-κB binding motif, as predicted, inhibited p50/p65 binding at each site (compare lanes 2–5 with lanes 7–10 and lanes 12–15 of Figure 3C). As expected, mutation of both NF-κB sites (κB I+II-Mut) resulted in abrogation of p50/p65 binding to the LTR. However, recombinant NFAT5 binding to the single and the double NF-κB mutant LTRs was not impaired and indeed appeared enhanced (compare lanes 2–5 to lanes 7–20 of Figure 3D). Thus, binding of NFAT5 or NF-κB p50/p65 to their respective motifs can be specifically disrupted within an overlapping or shared binding site. NF-κB and NFAT5 binding play independent activating roles in MTb-mediated induction of HIV-1 replication We next examined the functional impact of specifically disrupting NF-κB or NFAT5 binding on regulation of HIV-1 replication by infecting bulk PBMC from four normal donors with the wild-type or mutant HIV-1Lai/Bal-env molecular clones. After overnight infection, cells were co-infected with MTb (CDC1551) or mock-infected and free virus levels were detected by measuring p24 levels in culture supernatants at days 4, 7, and 12 post-HIV-1 infection. Replication of each mutant virus was reduced in comparison to wild-type virus in mock- and MTb-co-infected cells at all timepoints examined (Figures 4A–4B). As depicted in the histograms displayed in Figure 4C, at day 12 post-HIV-1 infection in MTb-co-infected cells there was a significant reduction in levels of HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, and HIV-1Lai/Bal-env -N5-Mut in comparison to wild-type virus (Figure 4C). In the absence of MTb co-infection, p24 levels were also significantly lower in the PBMC cultures infected with HIV-1Lai/Bal-env–κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut and HIV-1Lai/Bal-env-N5-Mut, but not HIV-1Lai/Bal-env-κB I-Mut, in comparison to wild-type virus (Figure 4C). None of the mutations completely abolished viral induction by MTb co-infection. However, the replication of each mutant virus was impaired to a more significant extent in the context of MTb co-infection, consistent with important roles for both NF-κB and NFAT5 in MTb-induced HIV-1 replication. 10.1371/journal.ppat.1002620.g004 Figure 4 MTb-induced HIV-1 replication in PBMC depends on binding of NFAT5 and NF-κB to the LTR. Specific disruption of the NF-κB or NFAT5 binding sites in the HIV-1 subtype B LTR inhibits R5-tropic virus replication in human PBMC after co-infection with MTb. PBMC from four normal donors were infected with 1000 TCID50 HIV-1Lai/Bal-env-WT or the mutants HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, or HIV-1Lai/Bal-env-N5-Mut and either; (A) left untreated, or (B) co-infected with MTb isolate CDC1551 (10∶1 cells∶bacilli). Viral p24 levels in culture supernatants were measured at days 4, 7 and 12 post-infection. (C) Histograms show viral p24 levels at day 12 in the MTb uninfected (grey bars) and MTb co-infected (black bars) PBMC cultures. Replication of the mutant viruses was compared to wild-type virus replication under the same experimental conditions (without and with MTb co-infection, respectively). *, p<0.05; **, p<0.01, as compared to HIV-1Lai/Bal-Env-WT. To specifically examine the functional impact of NF-κB and NFAT5 binding site usage in MDM, which are efficiently infected by both pathogens, we next isolated MDM from four normal donors and infected these cells with 1000 TCID50 of wild-type HIV-1Lai/Bal-env or with the mutant viral clones HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, or HIV-1Lai/Bal-env-N5-Mut. After overnight incubation, cell cultures were co-infected with MTb (CDC1551) or left infected with virus alone. Supernatants were collected at days 4, 7, and 12 post-HIV-1 infection and virus replication was measured. As shown in Figures 5A and 5C, in the absence of MTb co-infection, replication of mutant viral molecular clones (HIV-1Lai/Bal-env-κB I-Mut (p<0.05), HIV-1Lai/Bal-env-κB II-Mut (p<0.01), and HIV-1Lai/Bal-env-κB I+II-Mut (p<0.01) as well as HIV-1Lai/Bal-env-N5-Mut (p<0.01) were significantly reduced in comparison to the wild-type virus (Figures 5A–5C). 10.1371/journal.ppat.1002620.g005 Figure 5 Disruption of NF-κB or NFAT5 sites in the LTR inhibits MTb-induced HIV-1 replication in MDM. MDM from four normal donors were infected with 1000 TCID50 of HIV-1Lai/Bal-env–WT or the mutants HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, or HIV-1Lai/Bal-env-N5-Mut, and were either (A) left infected with virus alone or (B) co-infected with MTb CDC1551 (1∶1 MDM∶bacilli). Viral p24 levels in the culture supernatants were measured at days 4, 7 and 12 post-infection. Presentation of viral p24 levels at day 12 in cultures infected with (C) HIV-1 alone or (D) co-infected with MTb are shown as histograms. (*, p<0.05; **, p<0.01, as compared to HIV-1Lai/Bal-env–WT). When the cultures were co-infected with MTb, p24 levels increased when the cells were infected with either the wild-type or, to lower levels, with the κB-I mutant virus (Figure 5B). However, p24 levels were significantly inhibited in cells infected with HIV-1Lai/Bal-env -N5-Mut (p<0.01) (Figure 5D). Moreover, p24 levels were significantly lower at day 12 in cultures infected with HIV-1Lai/Bal-env-κB II-Mut and HIV-1Lai/Bal-env-κB I+II-Mut as compared to infection of cells with wild-type virus (Figure 5D). Notably, although replication of the mutant virus with the single, proximal NF-κB binding site mutation in the shared NFAT5/NF-κB element (HIV-1Lai/Bal-env-κB I-Mut) was diminished, this was not significant at day 12 (Figures 5B–5D). We note that overall p24 levels were noticeably lower in the cultures co-infected with MTb compared with those infected with HIV-1 alone. This is consistent with previous observations showing that MTb infection of human primary macrophage cultures ex vivo suppresses HIV-1 infection due to chemokine synthesis and the enhanced expression of cellular inhibitory factors [48]–[50]. Given that NF-κB is efficiently activated in primary MDM in response to TLR agonists [51] and as we have shown in Figure 2, NFAT5 gene expression is also induced by TLR agonists, MDM are a suitable experimental system to analyze the effect of NF-κB versus NFAT5 binding site mutations on virus replication in isolated MDM in the absence or presence of MTb co-infection. Taken together, the results from PBMC and MDM co-infection experiments demonstrate that the conserved NFAT5 binding site plays as important a transcriptional role in LTR regulation by MTb as do the NF-κB sites. NFAT5 is important for enhancement of HIV-1 subtype C replication in response to MTb infection As shown in Figure 1, the subtype C LTR was the most active of the HIV-1 LTR subtypes in the reporter assays. Subtype C LTRs generally have three functional NF-κB sites in their LTRs, and subtype C is the predominant viral subtype in the African and Asian HIV-1 epidemics where MTb co-infection is extremely common [1], [2], [28], [52]. We thus next extended our analysis of the role of NFAT5 in MTb/HIV-1 co-infection to a subtype C isolate. We first constructed an infectious molecular clone of the HIV-1 subtype C primary isolate HIV-198IN22, which has two NFAT5 binding sites in its LTR and three NF-κB sites (Figure 6A). We disrupted the two NFAT5 binding sites by changing the TT of each site to CC to create a mutant virus that we named HIV-198IN22-N5-Mut. Bulk PBMC from four normal donors were infected overnight with 1000 TCID50 of wild-type HIV-198IN22 or HIV-198IN22-N5-Mut and the cultures were then co-infected with MTb CDC1551 or left infected with virus alone. At day 11 post-viral infection, wild-type HIV-198IN22 replication was greater, but not significantly so, as compared to replication of HIV-198IN22 NFAT5-Mut (Figure 6B). However, in the context of MTb co-infection, wild-type HIV-198IN22 replication was significantly increased (p<0.05) at day 11 over HIV-198IN22 NFAT5-Mut replication (Figure 6C), indicating that the absence of NFAT5 binding sites was particularly detrimental to virus replication in the presence of MTb co-infection. Thus, even when three functional NF-κB binding sites are present in the LTR, as in the HIV-1 subtype C infectious clone studied here, disruption of NFAT5 binding to the LTR impairs virus replication in response to MTb co-infection. 10.1371/journal.ppat.1002620.g006 Figure 6 Disruption of the NFAT5 site in HIV-1 subtype C significantly impairs MTb-induced viral replication. (A) Mutation of the NFAT5 binding site in an HIV-1 subtype C infectious molecular clone. NFAT5 binding site mutations were introduced into the LTR of subtype C HIV-198IN22-WT. The NFAT5 binding site mutant (HIV-198IN22 N5-Mut) LTR sequence is shown alongside that of HIV-198IN22-WT. The HIV-198IN22 isolate analyzed here contains three NF-κB and two NFAT5 binding sites. The unique 3′ terminal adenine, which is important for NFAT5 binding to its site, is shown in blocks. Mutations were introduced into the 3′ LTR of the HIV-198IN22-WT proviral sequence. PBMC from four normal donors were infected with 1000 TCID50 of HIV-198IN22–WT or HIV-198IN22-N5-Mut. Cells were then (B) left infected with virus alone or (C) co-infected with MTb strain CDC1551 (10∶1 cells∶bacilli). Culture supernatants were collected at days 3, 7 and 11 post-infection and viral p24 levels were measured. Replication of HIV-198IN22-N5-Mut was significantly reduced at day 11 post-infection in comparison to HIV-198IN22–WT (*, p<0.05) in the presence of MTb co-infection. MTb induces NFAT5 gene expression via the MyD88-dependent signaling cascade
T22398 22078-22152 Sentence denotes (A) MTb but not HIV-1 increases NFAT5 mRNA synthesis in human macrophages.
T139 22078-22152 Sentence denotes (A) MTb but not HIV-1 increases NFAT5 mRNA synthesis in human macrophages.
T22399 22153-22214 Sentence denotes MDM were isolated from PBMC obtained from four normal donors.
T140 22153-22214 Sentence denotes MDM were isolated from PBMC obtained from four normal donors.
T22400 22215-22395 Sentence denotes The cells were incubated for 24 or 48 hours with heat-inactivated or live R5-tropic HIV-1 subtype B, C, or E isolates, live MTb strain CDC1551 (1∶1 MDM∶bacilli), or left untreated.
T141 22215-22395 Sentence denotes The cells were incubated for 24 or 48 hours with heat-inactivated or live R5-tropic HIV-1 subtype B, C, or E isolates, live MTb strain CDC1551 (1∶1 MDM∶bacilli), or left untreated.
T22401 22396-22854 Sentence denotes Quantitative real-time PCR analysis of NFAT5 mRNA expression levels revealed that the low, constitutively present level of NFAT5 mRNA expressed in untreated cells was not affected by exposure to live or inactivated HIV-1, but was significantly increased at both 24 (*, p<0.05) and 48 (**, p<0.01) hours following infection with MTb. (B) NFAT5-specific siRNA effectively suppresses NFAT5 expression in MDM in both the absence and presence of MTb co-infection.
T142 22396-22854 Sentence denotes Quantitative real-time PCR analysis of NFAT5 mRNA expression levels revealed that the low, constitutively present level of NFAT5 mRNA expressed in untreated cells was not affected by exposure to live or inactivated HIV-1, but was significantly increased at both 24 (*, p<0.05) and 48 (**, p<0.01) hours following infection with MTb. (B) NFAT5-specific siRNA effectively suppresses NFAT5 expression in MDM in both the absence and presence of MTb co-infection.
T22402 22855-23059 Sentence denotes MDM (1×106 cells/well in a 6-well plate) from four normal donors were transfected with siRNA specific for NFAT5 or, as a control, GFP, and were then infected with MTb (1∶1 MDM∶bacilli) or left uninfected.
T143 22855-23059 Sentence denotes MDM (1×106 cells/well in a 6-well plate) from four normal donors were transfected with siRNA specific for NFAT5 or, as a control, GFP, and were then infected with MTb (1∶1 MDM∶bacilli) or left uninfected.
T22403 23060-23206 Sentence denotes In the cells transfected with control siRNA, MTb infection significantly increased NFAT5 expression in comparison to uninfected cells (open bars).
T144 23060-23206 Sentence denotes In the cells transfected with control siRNA, MTb infection significantly increased NFAT5 expression in comparison to uninfected cells (open bars).
T22404 23207-23528 Sentence denotes When MDM were transfected with NFAT5-specific siRNA, both constitutively expressed (p = 0.048) and MTb-induced (p = 0.021) NFAT5 mRNA were significantly suppressed (grey bars). (C) HIV-1 subtype B replication is significantly suppressed in the presence of MTb co-infection when NFAT5 expression is abrogated in human MDM.
T145 23207-23528 Sentence denotes When MDM were transfected with NFAT5-specific siRNA, both constitutively expressed (p = 0.048) and MTb-induced (p = 0.021) NFAT5 mRNA were significantly suppressed (grey bars). (C) HIV-1 subtype B replication is significantly suppressed in the presence of MTb co-infection when NFAT5 expression is abrogated in human MDM.
T22405 23529-23632 Sentence denotes MDM from four normal donors were transfected with NFAT5-specific and control siRNAs as described in 2B.
T146 23529-23632 Sentence denotes MDM from four normal donors were transfected with NFAT5-specific and control siRNAs as described in 2B.
T22406 23633-23722 Sentence denotes Cells were then infected with HIV-1Lai/Bal-env followed by co-infection with MTb CDC1551.
T147 23633-23722 Sentence denotes Cells were then infected with HIV-1Lai/Bal-env followed by co-infection with MTb CDC1551.
T22407 23723-23790 Sentence denotes Virus replication was measured at days 6, 9, and 12 post-infection.
T148 23723-23790 Sentence denotes Virus replication was measured at days 6, 9, and 12 post-infection.
T22408 23791-23959 Sentence denotes At day 12, virus replication was significantly reduced in cells transfected with NFAT5-specific siRNA in comparison to cells transfected with control siRNA (*, p<0.05).
T149 23791-23959 Sentence denotes At day 12, virus replication was significantly reduced in cells transfected with NFAT5-specific siRNA in comparison to cells transfected with control siRNA (*, p<0.05).
T22409 23960-24122 Sentence denotes We monitored the morphology of cells within the infected cultures and did not observe noticeable macrophage necrosis even at the final timepoint of viral harvest.
T150 23960-24122 Sentence denotes We monitored the morphology of cells within the infected cultures and did not observe noticeable macrophage necrosis even at the final timepoint of viral harvest.
T8851 24124-24191 Sentence denotes RNAi directed against NFAT5 inhibits replication of subtype B HIV-1
T151 24124-24191 Sentence denotes RNAi directed against NFAT5 inhibits replication of subtype B HIV-1
T8852 24192-24471 Sentence denotes To extend the results we obtained in the reporter assays to a physiological TB/HIV co-infection model, we next tested the effect of siRNA-mediated ablation of NFAT5 mRNA levels in MDM co-infected with a subtype B HIV-1Lai infectious molecular clone and a clinical isolate of MTb.
T152 24192-24471 Sentence denotes To extend the results we obtained in the reporter assays to a physiological TB/HIV co-infection model, we next tested the effect of siRNA-mediated ablation of NFAT5 mRNA levels in MDM co-infected with a subtype B HIV-1Lai infectious molecular clone and a clinical isolate of MTb.
T8853 24472-24658 Sentence denotes To perform this experiment, we first constructed an HIV-1Lai clone bearing the CCR5-tropic envelope region of HIV-1Bal (HIV-1Lai/Bal-env) so that it would efficiently infect primary MDM.
T153 24472-24658 Sentence denotes To perform this experiment, we first constructed an HIV-1Lai clone bearing the CCR5-tropic envelope region of HIV-1Bal (HIV-1Lai/Bal-env) so that it would efficiently infect primary MDM.
T8854 24659-24934 Sentence denotes We used this approach to ensure that our analysis of the roles of NF-κB and NFAT5 in MTb-induced HIV-1 replication could be interpreted in the proper context of previous research findings that examined LTR regulation in the context of full-length viral replication [41]–[46].
T154 24659-24934 Sentence denotes We used this approach to ensure that our analysis of the roles of NF-κB and NFAT5 in MTb-induced HIV-1 replication could be interpreted in the proper context of previous research findings that examined LTR regulation in the context of full-length viral replication [41]–[46].
T8855 24935-25192 Sentence denotes The HIV-1Lai/Bal-Env infectious clone is isogenic for the entire sequence of the parental HIV-1Lai infectious clone except for the substitution of the HIV-1Bal envelope co-receptor binding region in place of the HIV-1Lai envelope co-receptor binding region.
T155 24935-25192 Sentence denotes The HIV-1Lai/Bal-Env infectious clone is isogenic for the entire sequence of the parental HIV-1Lai infectious clone except for the substitution of the HIV-1Bal envelope co-receptor binding region in place of the HIV-1Lai envelope co-receptor binding region.
T8856 25193-25398 Sentence denotes We note that we confirmed that HIV-1Lai/Bal-Env grew in PBMC at a similar rate to wild-type HIV-1Bal, indicating proper co-receptor engagement and internalization of this infectious clone (data not shown).
T156 25193-25398 Sentence denotes We note that we confirmed that HIV-1Lai/Bal-Env grew in PBMC at a similar rate to wild-type HIV-1Bal, indicating proper co-receptor engagement and internalization of this infectious clone (data not shown).
T8857 25399-25523 Sentence denotes Next, we knocked down NFAT5 mRNA levels in MDM using an siRNA that suppresses both NFAT5 mRNA and NFAT5 protein levels [31].
T157 25399-25523 Sentence denotes Next, we knocked down NFAT5 mRNA levels in MDM using an siRNA that suppresses both NFAT5 mRNA and NFAT5 protein levels [31].
T8858 25524-25783 Sentence denotes As shown in Figure 2, transfection of the siRNA specific for NFAT5 reduces NFAT5 mRNA levels in both MTb-uninfected (p = 0.048) and MTb-infected (p = 0.021) MDM as compared to transfection of control GFP siRNA into MTb-uninfected or -infected MDM (Figure 2B).
T158 25524-25783 Sentence denotes As shown in Figure 2, transfection of the siRNA specific for NFAT5 reduces NFAT5 mRNA levels in both MTb-uninfected (p = 0.048) and MTb-infected (p = 0.021) MDM as compared to transfection of control GFP siRNA into MTb-uninfected or -infected MDM (Figure 2B).
T8859 25784-26031 Sentence denotes We note that although siRNA normally is effective for 48–72 hours in cell lines that divide rapidly, in human MDM, which are non-dividing cells, siRNA to host factors remains detectable and functional up to at least 15 days post-transfection [47].
T159 25784-26031 Sentence denotes We note that although siRNA normally is effective for 48–72 hours in cell lines that divide rapidly, in human MDM, which are non-dividing cells, siRNA to host factors remains detectable and functional up to at least 15 days post-transfection [47].
T8860 26032-26202 Sentence denotes MDM in which NFAT5 expression had been inhibited with NFAT5 siRNA or that were transfected with control GFP siRNA were then infected with 1000 TCID50 of HIV-1Lai/Bal-env.
T160 26032-26202 Sentence denotes MDM in which NFAT5 expression had been inhibited with NFAT5 siRNA or that were transfected with control GFP siRNA were then infected with 1000 TCID50 of HIV-1Lai/Bal-env.
T8861 26203-26305 Sentence denotes After overnight virus infection, the cells were then co-infected with the MTb clinical strain CDC1551.
T161 26203-26305 Sentence denotes After overnight virus infection, the cells were then co-infected with the MTb clinical strain CDC1551.
T8862 26306-26549 Sentence denotes Free virus levels were then measured in culture supernatants from MDM transfected with NFAT5-specific siRNA or GFP control siRNA at days 6, 9 and 12 post-HIV-1 infection to measure the impact of NFAT5 inhibition on MTb-induced HIV replication.
T162 26306-26549 Sentence denotes Free virus levels were then measured in culture supernatants from MDM transfected with NFAT5-specific siRNA or GFP control siRNA at days 6, 9 and 12 post-HIV-1 infection to measure the impact of NFAT5 inhibition on MTb-induced HIV replication.
T8863 26550-26800 Sentence denotes As shown in Figure 2C, HIV-1 replication was suppressed at days 6 and 9 post-infection in the NFAT5 siRNA-treated cells as compared to cells treated with control siRNA, and it was significantly inhibited by day 12 post-infection (p<0.05) (Figure 2C).
T163 26550-26800 Sentence denotes As shown in Figure 2C, HIV-1 replication was suppressed at days 6 and 9 post-infection in the NFAT5 siRNA-treated cells as compared to cells treated with control siRNA, and it was significantly inhibited by day 12 post-infection (p<0.05) (Figure 2C).
T8864 26801-26916 Sentence denotes Thus, knock down of NFAT5 expression significantly impairs HIV-1 subtype B replication in MDM co-infected with MTb.
T164 26801-26916 Sentence denotes Thus, knock down of NFAT5 expression significantly impairs HIV-1 subtype B replication in MDM co-infected with MTb.
T10446 26918-26988 Sentence denotes NF-κB and NFAT5 binding to the HIV-1 LTR can be specifically disrupted
T165 26918-26988 Sentence denotes NF-κB and NFAT5 binding to the HIV-1 LTR can be specifically disrupted
T10447 26989-27296 Sentence denotes To demonstrate that the impact of NFAT5 silencing on MTb-induced viral replication was a direct effect due to modulation of recruitment of NFAT5 to the HIV-1 LTR and not due to secondary, NFAT5-regulated effects, we set out to disrupt NFAT5 binding to the viral LTR in the context of HIV-1/MTb co-infection.
T166 26989-27296 Sentence denotes To demonstrate that the impact of NFAT5 silencing on MTb-induced viral replication was a direct effect due to modulation of recruitment of NFAT5 to the HIV-1 LTR and not due to secondary, NFAT5-regulated effects, we set out to disrupt NFAT5 binding to the viral LTR in the context of HIV-1/MTb co-infection.
T10448 27297-27458 Sentence denotes This series of experiments also allowed us to dissect the relative importance of NFAT5 and NF-κB binding to the HIV-1 LTR in MTb regulation of HIV-1 replication.
T167 27297-27458 Sentence denotes This series of experiments also allowed us to dissect the relative importance of NFAT5 and NF-κB binding to the HIV-1 LTR in MTb regulation of HIV-1 replication.
T10449 27459-27703 Sentence denotes To perform these experiments, we constructed a panel of infectious HIV-1Lai/Bal-env molecular clones where either the NFAT5 site was specifically disrupted or the two NF-κB binding sites were either individually or dually disrupted (Figure 3A).
T168 27459-27703 Sentence denotes To perform these experiments, we constructed a panel of infectious HIV-1Lai/Bal-env molecular clones where either the NFAT5 site was specifically disrupted or the two NF-κB binding sites were either individually or dually disrupted (Figure 3A).
T10450 27704-28041 Sentence denotes Specifically, we constructed a clone in which the NFAT5 binding site was mutated (named HIV-1Lai/Bal-env-N5-Mut) by changing the CC dinucleotide to TT in the core NFAT5 binding site and introduced substitution mutations into the NF-κB/NFAT5 shared binding element that we predicted would disrupt NF-κB binding but preserve NFAT5 binding.
T169 27704-28041 Sentence denotes Specifically, we constructed a clone in which the NFAT5 binding site was mutated (named HIV-1Lai/Bal-env-N5-Mut) by changing the CC dinucleotide to TT in the core NFAT5 binding site and introduced substitution mutations into the NF-κB/NFAT5 shared binding element that we predicted would disrupt NF-κB binding but preserve NFAT5 binding.
T10451 28042-28315 Sentence denotes Specifically, we mutated two guanines (GG) in the κB I site and changed them to thymine and adenine (TA) (HIV-1Lai/Bal-env-κB I-mut) and also introduced the same GG to TA change in the second, more distal NF-κB site (κB II) in HIV-1Lai/Bal-env (HIV-1Lai/Bal-env-κB II-Mut).
T170 28042-28315 Sentence denotes Specifically, we mutated two guanines (GG) in the κB I site and changed them to thymine and adenine (TA) (HIV-1Lai/Bal-env-κB I-mut) and also introduced the same GG to TA change in the second, more distal NF-κB site (κB II) in HIV-1Lai/Bal-env (HIV-1Lai/Bal-env-κB II-Mut).
T10452 28316-28499 Sentence denotes We also created a double NF-κB site mutant virus (HIV-1Lai/Bal-env-κB I+II-Mut) in order to test the impact of complete disruption of NF-κB binding to the HIV-1 LTR on MTb regulation.
T171 28316-28499 Sentence denotes We also created a double NF-κB site mutant virus (HIV-1Lai/Bal-env-κB I+II-Mut) in order to test the impact of complete disruption of NF-κB binding to the HIV-1 LTR on MTb regulation.
T172 28500-28626 Sentence denotes 10.1371/journal.ppat.1002620.g003 Figure 3 Specific disruption of NFAT5 or NF-κB binding sites in the LTR of HIV-1 subtype B.
T23761 28544-28626 Sentence denotes Specific disruption of NFAT5 or NF-κB binding sites in the LTR of HIV-1 subtype B.
T23762 28627-28703 Sentence denotes (A) Mutations introduced into a HIV-1Lai/Bal-env infectious molecular clone.
T173 28627-28703 Sentence denotes (A) Mutations introduced into a HIV-1Lai/Bal-env infectious molecular clone.
T23763 28704-28838 Sentence denotes NF-κB and NFAT5 binding site mutations were introduced into the LTR of the HIV-1Lai/Bal-env-wild type (WT) infectious molecular clone.
T174 28704-28838 Sentence denotes NF-κB and NFAT5 binding site mutations were introduced into the LTR of the HIV-1Lai/Bal-env-wild type (WT) infectious molecular clone.
T23764 28839-29067 Sentence denotes The NF-κB binding site mutants (HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut), and NFAT5 binding site mutant (HIV-1Lai/Bal-env-N5-Mut) are shown along with the HIV-1Lai/Bal-env-WT sequence.
T175 28839-29067 Sentence denotes The NF-κB binding site mutants (HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut), and NFAT5 binding site mutant (HIV-1Lai/Bal-env-N5-Mut) are shown along with the HIV-1Lai/Bal-env-WT sequence.
T23765 29068-29325 Sentence denotes Mutations were introduced into the 3′ LTR of the HIV-1Lai/Bal-env–WT proviral sequence. (B) Specific mutation of the NFAT5 binding site abolishes NFAT5 binding to the viral LTR but does not affect NF-κB p50/p65 binding to the overlapping NF-κB binding site.
T176 29068-29325 Sentence denotes Mutations were introduced into the 3′ LTR of the HIV-1Lai/Bal-env–WT proviral sequence. (B) Specific mutation of the NFAT5 binding site abolishes NFAT5 binding to the viral LTR but does not affect NF-κB p50/p65 binding to the overlapping NF-κB binding site.
T23766 29326-29639 Sentence denotes Quantitative DNaseI footprinting analysis is shown using HIV-1 LTR fragments (-262 to +4 nt relative to the transcription start site) from HIV-1Lai/Bal-env-WT and HIV-1Lai/Bal-env-N5-Mut and increasing concentrations of recombinant NF-κB (p50/p65) (25 ng, 100 ng, and 500 ng), or NFAT5 (10 ng, 50 ng, and 250 ng).
T177 29326-29639 Sentence denotes Quantitative DNaseI footprinting analysis is shown using HIV-1 LTR fragments (-262 to +4 nt relative to the transcription start site) from HIV-1Lai/Bal-env-WT and HIV-1Lai/Bal-env-N5-Mut and increasing concentrations of recombinant NF-κB (p50/p65) (25 ng, 100 ng, and 500 ng), or NFAT5 (10 ng, 50 ng, and 250 ng).
T23767 29640-29871 Sentence denotes The regions that are protected from DNase I cleavage by the binding of NF-κB and NFAT5 are indicated with a bars. (C) Specific disruption of the HIV-1 subtype B NF-κB binding sites effectively abrogates recombinant p50/p65 binding.
T178 29640-29871 Sentence denotes The regions that are protected from DNase I cleavage by the binding of NF-κB and NFAT5 are indicated with a bars. (C) Specific disruption of the HIV-1 subtype B NF-κB binding sites effectively abrogates recombinant p50/p65 binding.
T23768 29872-30152 Sentence denotes Quantitative DNaseI footprinting analysis is shown of nucleotides −262 to +4 from HIV-1Lai/Bal-env-WT, HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, and HIV-1Lai/Bal-env-κB I+II-Mut and increasing concentrations of recombinant NF-κB (p50/p65) (25 ng, 100 ng, and 500 ng).
T179 29872-30152 Sentence denotes Quantitative DNaseI footprinting analysis is shown of nucleotides −262 to +4 from HIV-1Lai/Bal-env-WT, HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, and HIV-1Lai/Bal-env-κB I+II-Mut and increasing concentrations of recombinant NF-κB (p50/p65) (25 ng, 100 ng, and 500 ng).
T23769 30153-30394 Sentence denotes The regions that are protected from DNase I cleavage by the binding of recombinant NF-κB are indicated with a bars. (D) Specific disruption of the HIV-1 subtype B NF-κB binding site does not inhibit but enhances NFAT5 binding to this region.
T180 30153-30394 Sentence denotes The regions that are protected from DNase I cleavage by the binding of recombinant NF-κB are indicated with a bars. (D) Specific disruption of the HIV-1 subtype B NF-κB binding site does not inhibit but enhances NFAT5 binding to this region.
T23770 30395-30664 Sentence denotes Quantitative DNaseI footprinting analysis is shown of nucleotides −262 to +4 from HIV-1Lai/Bal-env-WT, HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, and HIV-1Lai/Bal-env-κB I+II-Mut and increasing concentrations of recombinant NFAT5 (10 ng, 50 ng, and 250 ng).
T181 30395-30664 Sentence denotes Quantitative DNaseI footprinting analysis is shown of nucleotides −262 to +4 from HIV-1Lai/Bal-env-WT, HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, and HIV-1Lai/Bal-env-κB I+II-Mut and increasing concentrations of recombinant NFAT5 (10 ng, 50 ng, and 250 ng).
T23771 30665-30780 Sentence denotes The regions that are protected from DNase I cleavage by the binding of recombinant NFAT5 are indicated with a bars.
T182 30665-30780 Sentence denotes The regions that are protected from DNase I cleavage by the binding of recombinant NFAT5 are indicated with a bars.
T10453 30781-31072 Sentence denotes To determine the specificity of these substitutions upon NFAT5 and NF-κB p50/p65 binding to the LTR, we performed a quantitative DNase I footprinting analysis using the wild-type LTR or mutant LTRs in combination with increasing concentrations of recombinant NFAT5 or NF-κB p50/p65 proteins.
T183 30781-31072 Sentence denotes To determine the specificity of these substitutions upon NFAT5 and NF-κB p50/p65 binding to the LTR, we performed a quantitative DNase I footprinting analysis using the wild-type LTR or mutant LTRs in combination with increasing concentrations of recombinant NFAT5 or NF-κB p50/p65 proteins.
T10454 31073-31244 Sentence denotes As shown in Figure 3B, the N5-Mut LTR could not bind NFAT5 (compare lanes 12–15 and lanes 17–20), but p50/p65 binding was not impaired (compare lanes 2–5 with lanes 7–10).
T184 31073-31244 Sentence denotes As shown in Figure 3B, the N5-Mut LTR could not bind NFAT5 (compare lanes 12–15 and lanes 17–20), but p50/p65 binding was not impaired (compare lanes 2–5 with lanes 7–10).
T10455 31245-31442 Sentence denotes By contrast, changing the GG dinucleotide to TA in either NF-κB binding motif, as predicted, inhibited p50/p65 binding at each site (compare lanes 2–5 with lanes 7–10 and lanes 12–15 of Figure 3C).
T185 31245-31442 Sentence denotes By contrast, changing the GG dinucleotide to TA in either NF-κB binding motif, as predicted, inhibited p50/p65 binding at each site (compare lanes 2–5 with lanes 7–10 and lanes 12–15 of Figure 3C).
T10456 31443-31552 Sentence denotes As expected, mutation of both NF-κB sites (κB I+II-Mut) resulted in abrogation of p50/p65 binding to the LTR.
T186 31443-31552 Sentence denotes As expected, mutation of both NF-κB sites (κB I+II-Mut) resulted in abrogation of p50/p65 binding to the LTR.
T10457 31553-31728 Sentence denotes However, recombinant NFAT5 binding to the single and the double NF-κB mutant LTRs was not impaired and indeed appeared enhanced (compare lanes 2–5 to lanes 7–20 of Figure 3D).
T187 31553-31728 Sentence denotes However, recombinant NFAT5 binding to the single and the double NF-κB mutant LTRs was not impaired and indeed appeared enhanced (compare lanes 2–5 to lanes 7–20 of Figure 3D).
T10458 31729-31871 Sentence denotes Thus, binding of NFAT5 or NF-κB p50/p65 to their respective motifs can be specifically disrupted within an overlapping or shared binding site.
T188 31729-31871 Sentence denotes Thus, binding of NFAT5 or NF-κB p50/p65 to their respective motifs can be specifically disrupted within an overlapping or shared binding site.
T12297 31873-31977 Sentence denotes NF-κB and NFAT5 binding play independent activating roles in MTb-mediated induction of HIV-1 replication
T189 31873-31977 Sentence denotes NF-κB and NFAT5 binding play independent activating roles in MTb-mediated induction of HIV-1 replication
T12298 31978-32212 Sentence denotes We next examined the functional impact of specifically disrupting NF-κB or NFAT5 binding on regulation of HIV-1 replication by infecting bulk PBMC from four normal donors with the wild-type or mutant HIV-1Lai/Bal-env molecular clones.
T190 31978-32212 Sentence denotes We next examined the functional impact of specifically disrupting NF-κB or NFAT5 binding on regulation of HIV-1 replication by infecting bulk PBMC from four normal donors with the wild-type or mutant HIV-1Lai/Bal-env molecular clones.
T12299 32213-32425 Sentence denotes After overnight infection, cells were co-infected with MTb (CDC1551) or mock-infected and free virus levels were detected by measuring p24 levels in culture supernatants at days 4, 7, and 12 post-HIV-1 infection.
T191 32213-32425 Sentence denotes After overnight infection, cells were co-infected with MTb (CDC1551) or mock-infected and free virus levels were detected by measuring p24 levels in culture supernatants at days 4, 7, and 12 post-HIV-1 infection.
T12300 32426-32582 Sentence denotes Replication of each mutant virus was reduced in comparison to wild-type virus in mock- and MTb-co-infected cells at all timepoints examined (Figures 4A–4B).
T192 32426-32582 Sentence denotes Replication of each mutant virus was reduced in comparison to wild-type virus in mock- and MTb-co-infected cells at all timepoints examined (Figures 4A–4B).
T12301 32583-32899 Sentence denotes As depicted in the histograms displayed in Figure 4C, at day 12 post-HIV-1 infection in MTb-co-infected cells there was a significant reduction in levels of HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, and HIV-1Lai/Bal-env -N5-Mut in comparison to wild-type virus (Figure 4C).
T193 32583-32899 Sentence denotes As depicted in the histograms displayed in Figure 4C, at day 12 post-HIV-1 infection in MTb-co-infected cells there was a significant reduction in levels of HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, and HIV-1Lai/Bal-env -N5-Mut in comparison to wild-type virus (Figure 4C).
T12302 32900-33178 Sentence denotes In the absence of MTb co-infection, p24 levels were also significantly lower in the PBMC cultures infected with HIV-1Lai/Bal-env–κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut and HIV-1Lai/Bal-env-N5-Mut, but not HIV-1Lai/Bal-env-κB I-Mut, in comparison to wild-type virus (Figure 4C).
T194 32900-33178 Sentence denotes In the absence of MTb co-infection, p24 levels were also significantly lower in the PBMC cultures infected with HIV-1Lai/Bal-env–κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut and HIV-1Lai/Bal-env-N5-Mut, but not HIV-1Lai/Bal-env-κB I-Mut, in comparison to wild-type virus (Figure 4C).
T12303 33179-33258 Sentence denotes None of the mutations completely abolished viral induction by MTb co-infection.
T195 33179-33258 Sentence denotes None of the mutations completely abolished viral induction by MTb co-infection.
T12304 33259-33473 Sentence denotes However, the replication of each mutant virus was impaired to a more significant extent in the context of MTb co-infection, consistent with important roles for both NF-κB and NFAT5 in MTb-induced HIV-1 replication.
T196 33259-33473 Sentence denotes However, the replication of each mutant virus was impaired to a more significant extent in the context of MTb co-infection, consistent with important roles for both NF-κB and NFAT5 in MTb-induced HIV-1 replication.
T197 33474-33605 Sentence denotes 10.1371/journal.ppat.1002620.g004 Figure 4 MTb-induced HIV-1 replication in PBMC depends on binding of NFAT5 and NF-κB to the LTR.
T25271 33518-33605 Sentence denotes MTb-induced HIV-1 replication in PBMC depends on binding of NFAT5 and NF-κB to the LTR.
T25272 33606-33768 Sentence denotes Specific disruption of the NF-κB or NFAT5 binding sites in the HIV-1 subtype B LTR inhibits R5-tropic virus replication in human PBMC after co-infection with MTb.
T198 33606-33768 Sentence denotes Specific disruption of the NF-κB or NFAT5 binding sites in the HIV-1 subtype B LTR inhibits R5-tropic virus replication in human PBMC after co-infection with MTb.
T25273 33769-34073 Sentence denotes PBMC from four normal donors were infected with 1000 TCID50 HIV-1Lai/Bal-env-WT or the mutants HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, or HIV-1Lai/Bal-env-N5-Mut and either; (A) left untreated, or (B) co-infected with MTb isolate CDC1551 (10∶1 cells∶bacilli).
T199 33769-34073 Sentence denotes PBMC from four normal donors were infected with 1000 TCID50 HIV-1Lai/Bal-env-WT or the mutants HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, or HIV-1Lai/Bal-env-N5-Mut and either; (A) left untreated, or (B) co-infected with MTb isolate CDC1551 (10∶1 cells∶bacilli).
T25274 34074-34293 Sentence denotes Viral p24 levels in culture supernatants were measured at days 4, 7 and 12 post-infection. (C) Histograms show viral p24 levels at day 12 in the MTb uninfected (grey bars) and MTb co-infected (black bars) PBMC cultures.
T200 34074-34293 Sentence denotes Viral p24 levels in culture supernatants were measured at days 4, 7 and 12 post-infection. (C) Histograms show viral p24 levels at day 12 in the MTb uninfected (grey bars) and MTb co-infected (black bars) PBMC cultures.
T25275 34294-34520 Sentence denotes Replication of the mutant viruses was compared to wild-type virus replication under the same experimental conditions (without and with MTb co-infection, respectively). *, p<0.05; **, p<0.01, as compared to HIV-1Lai/Bal-Env-WT.
T201 34294-34520 Sentence denotes Replication of the mutant viruses was compared to wild-type virus replication under the same experimental conditions (without and with MTb co-infection, respectively). *, p<0.05; **, p<0.01, as compared to HIV-1Lai/Bal-Env-WT.
T202 34521-34924 Sentence denotes To specifically examine the functional impact of NF-κB and NFAT5 binding site usage in MDM, which are efficiently infected by both pathogens, we next isolated MDM from four normal donors and infected these cells with 1000 TCID50 of wild-type HIV-1Lai/Bal-env or with the mutant viral clones HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, or HIV-1Lai/Bal-env-N5-Mut.
T12305 34521-35037 Sentence denotes To specifically examine the functional impact of NF-κB and NFAT5 binding site usage in MDM, which are efficiently infected by both pathogens, we next isolated MDM from four normal donors and infected these cells with 1000 TCID50 of wild-type HIV-1Lai/Bal-env or with the mutant viral clones HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, or HIV-1Lai/Bal-env-N5-Mut. After overnight incubation, cell cultures were co-infected with MTb (CDC1551) or left infected with virus alone.
T203 34925-35037 Sentence denotes After overnight incubation, cell cultures were co-infected with MTb (CDC1551) or left infected with virus alone.
T12306 35038-35143 Sentence denotes Supernatants were collected at days 4, 7, and 12 post-HIV-1 infection and virus replication was measured.
T204 35038-35143 Sentence denotes Supernatants were collected at days 4, 7, and 12 post-HIV-1 infection and virus replication was measured.
T12307 35144-35496 Sentence denotes As shown in Figures 5A and 5C, in the absence of MTb co-infection, replication of mutant viral molecular clones (HIV-1Lai/Bal-env-κB I-Mut (p<0.05), HIV-1Lai/Bal-env-κB II-Mut (p<0.01), and HIV-1Lai/Bal-env-κB I+II-Mut (p<0.01) as well as HIV-1Lai/Bal-env-N5-Mut (p<0.01) were significantly reduced in comparison to the wild-type virus (Figures 5A–5C).
T205 35144-35496 Sentence denotes As shown in Figures 5A and 5C, in the absence of MTb co-infection, replication of mutant viral molecular clones (HIV-1Lai/Bal-env-κB I-Mut (p<0.05), HIV-1Lai/Bal-env-κB II-Mut (p<0.01), and HIV-1Lai/Bal-env-κB I+II-Mut (p<0.01) as well as HIV-1Lai/Bal-env-N5-Mut (p<0.01) were significantly reduced in comparison to the wild-type virus (Figures 5A–5C).
T206 35497-35633 Sentence denotes 10.1371/journal.ppat.1002620.g005 Figure 5 Disruption of NF-κB or NFAT5 sites in the LTR inhibits MTb-induced HIV-1 replication in MDM.
T25855 35541-35633 Sentence denotes Disruption of NF-κB or NFAT5 sites in the LTR inhibits MTb-induced HIV-1 replication in MDM.
T25856 35634-35949 Sentence denotes MDM from four normal donors were infected with 1000 TCID50 of HIV-1Lai/Bal-env–WT or the mutants HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, or HIV-1Lai/Bal-env-N5-Mut, and were either (A) left infected with virus alone or (B) co-infected with MTb CDC1551 (1∶1 MDM∶bacilli).
T207 35634-35949 Sentence denotes MDM from four normal donors were infected with 1000 TCID50 of HIV-1Lai/Bal-env–WT or the mutants HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, or HIV-1Lai/Bal-env-N5-Mut, and were either (A) left infected with virus alone or (B) co-infected with MTb CDC1551 (1∶1 MDM∶bacilli).
T25857 35950-36044 Sentence denotes Viral p24 levels in the culture supernatants were measured at days 4, 7 and 12 post-infection.
T208 35950-36044 Sentence denotes Viral p24 levels in the culture supernatants were measured at days 4, 7 and 12 post-infection.
T25858 36045-36243 Sentence denotes Presentation of viral p24 levels at day 12 in cultures infected with (C) HIV-1 alone or (D) co-infected with MTb are shown as histograms. (*, p<0.05; **, p<0.01, as compared to HIV-1Lai/Bal-env–WT).
T209 36045-36243 Sentence denotes Presentation of viral p24 levels at day 12 in cultures infected with (C) HIV-1 alone or (D) co-infected with MTb are shown as histograms. (*, p<0.05; **, p<0.01, as compared to HIV-1Lai/Bal-env–WT).
T12308 36244-36425 Sentence denotes When the cultures were co-infected with MTb, p24 levels increased when the cells were infected with either the wild-type or, to lower levels, with the κB-I mutant virus (Figure 5B).
T210 36244-36425 Sentence denotes When the cultures were co-infected with MTb, p24 levels increased when the cells were infected with either the wild-type or, to lower levels, with the κB-I mutant virus (Figure 5B).
T12309 36426-36544 Sentence denotes However, p24 levels were significantly inhibited in cells infected with HIV-1Lai/Bal-env -N5-Mut (p<0.01) (Figure 5D).
T211 36426-36544 Sentence denotes However, p24 levels were significantly inhibited in cells infected with HIV-1Lai/Bal-env -N5-Mut (p<0.01) (Figure 5D).
T12310 36545-36754 Sentence denotes Moreover, p24 levels were significantly lower at day 12 in cultures infected with HIV-1Lai/Bal-env-κB II-Mut and HIV-1Lai/Bal-env-κB I+II-Mut as compared to infection of cells with wild-type virus (Figure 5D).
T212 36545-36754 Sentence denotes Moreover, p24 levels were significantly lower at day 12 in cultures infected with HIV-1Lai/Bal-env-κB II-Mut and HIV-1Lai/Bal-env-κB I+II-Mut as compared to infection of cells with wild-type virus (Figure 5D).
T12311 36755-36988 Sentence denotes Notably, although replication of the mutant virus with the single, proximal NF-κB binding site mutation in the shared NFAT5/NF-κB element (HIV-1Lai/Bal-env-κB I-Mut) was diminished, this was not significant at day 12 (Figures 5B–5D).
T213 36755-36988 Sentence denotes Notably, although replication of the mutant virus with the single, proximal NF-κB binding site mutation in the shared NFAT5/NF-κB element (HIV-1Lai/Bal-env-κB I-Mut) was diminished, this was not significant at day 12 (Figures 5B–5D).
T12312 36989-37126 Sentence denotes We note that overall p24 levels were noticeably lower in the cultures co-infected with MTb compared with those infected with HIV-1 alone.
T214 36989-37126 Sentence denotes We note that overall p24 levels were noticeably lower in the cultures co-infected with MTb compared with those infected with HIV-1 alone.
T12313 37127-37368 Sentence denotes This is consistent with previous observations showing that MTb infection of human primary macrophage cultures ex vivo suppresses HIV-1 infection due to chemokine synthesis and the enhanced expression of cellular inhibitory factors [48]–[50].
T215 37127-37368 Sentence denotes This is consistent with previous observations showing that MTb infection of human primary macrophage cultures ex vivo suppresses HIV-1 infection due to chemokine synthesis and the enhanced expression of cellular inhibitory factors [48]–[50].
T12314 37369-37738 Sentence denotes Given that NF-κB is efficiently activated in primary MDM in response to TLR agonists [51] and as we have shown in Figure 2, NFAT5 gene expression is also induced by TLR agonists, MDM are a suitable experimental system to analyze the effect of NF-κB versus NFAT5 binding site mutations on virus replication in isolated MDM in the absence or presence of MTb co-infection.
T216 37369-37738 Sentence denotes Given that NF-κB is efficiently activated in primary MDM in response to TLR agonists [51] and as we have shown in Figure 2, NFAT5 gene expression is also induced by TLR agonists, MDM are a suitable experimental system to analyze the effect of NF-κB versus NFAT5 binding site mutations on virus replication in isolated MDM in the absence or presence of MTb co-infection.
T12315 37739-37949 Sentence denotes Taken together, the results from PBMC and MDM co-infection experiments demonstrate that the conserved NFAT5 binding site plays as important a transcriptional role in LTR regulation by MTb as do the NF-κB sites.
T217 37739-37949 Sentence denotes Taken together, the results from PBMC and MDM co-infection experiments demonstrate that the conserved NFAT5 binding site plays as important a transcriptional role in LTR regulation by MTb as do the NF-κB sites.
T13706 37951-38045 Sentence denotes NFAT5 is important for enhancement of HIV-1 subtype C replication in response to MTb infection
T218 37951-38045 Sentence denotes NFAT5 is important for enhancement of HIV-1 subtype C replication in response to MTb infection
T13707 38046-38155 Sentence denotes As shown in Figure 1, the subtype C LTR was the most active of the HIV-1 LTR subtypes in the reporter assays.
T219 38046-38155 Sentence denotes As shown in Figure 1, the subtype C LTR was the most active of the HIV-1 LTR subtypes in the reporter assays.
T13708 38156-38382 Sentence denotes Subtype C LTRs generally have three functional NF-κB sites in their LTRs, and subtype C is the predominant viral subtype in the African and Asian HIV-1 epidemics where MTb co-infection is extremely common [1], [2], [28], [52].
T220 38156-38382 Sentence denotes Subtype C LTRs generally have three functional NF-κB sites in their LTRs, and subtype C is the predominant viral subtype in the African and Asian HIV-1 epidemics where MTb co-infection is extremely common [1], [2], [28], [52].
T13709 38383-38488 Sentence denotes We thus next extended our analysis of the role of NFAT5 in MTb/HIV-1 co-infection to a subtype C isolate.
T221 38383-38488 Sentence denotes We thus next extended our analysis of the role of NFAT5 in MTb/HIV-1 co-infection to a subtype C isolate.
T13710 38489-38671 Sentence denotes We first constructed an infectious molecular clone of the HIV-1 subtype C primary isolate HIV-198IN22, which has two NFAT5 binding sites in its LTR and three NF-κB sites (Figure 6A).
T222 38489-38671 Sentence denotes We first constructed an infectious molecular clone of the HIV-1 subtype C primary isolate HIV-198IN22, which has two NFAT5 binding sites in its LTR and three NF-κB sites (Figure 6A).
T223 38672-38809 Sentence denotes We disrupted the two NFAT5 binding sites by changing the TT of each site to CC to create a mutant virus that we named HIV-198IN22-N5-Mut.
T13711 38672-39022 Sentence denotes We disrupted the two NFAT5 binding sites by changing the TT of each site to CC to create a mutant virus that we named HIV-198IN22-N5-Mut. Bulk PBMC from four normal donors were infected overnight with 1000 TCID50 of wild-type HIV-198IN22 or HIV-198IN22-N5-Mut and the cultures were then co-infected with MTb CDC1551 or left infected with virus alone.
T224 38810-39022 Sentence denotes Bulk PBMC from four normal donors were infected overnight with 1000 TCID50 of wild-type HIV-198IN22 or HIV-198IN22-N5-Mut and the cultures were then co-infected with MTb CDC1551 or left infected with virus alone.
T13712 39023-39192 Sentence denotes At day 11 post-viral infection, wild-type HIV-198IN22 replication was greater, but not significantly so, as compared to replication of HIV-198IN22 NFAT5-Mut (Figure 6B).
T225 39023-39192 Sentence denotes At day 11 post-viral infection, wild-type HIV-198IN22 replication was greater, but not significantly so, as compared to replication of HIV-198IN22 NFAT5-Mut (Figure 6B).
T13713 39193-39508 Sentence denotes However, in the context of MTb co-infection, wild-type HIV-198IN22 replication was significantly increased (p<0.05) at day 11 over HIV-198IN22 NFAT5-Mut replication (Figure 6C), indicating that the absence of NFAT5 binding sites was particularly detrimental to virus replication in the presence of MTb co-infection.
T226 39193-39508 Sentence denotes However, in the context of MTb co-infection, wild-type HIV-198IN22 replication was significantly increased (p<0.05) at day 11 over HIV-198IN22 NFAT5-Mut replication (Figure 6C), indicating that the absence of NFAT5 binding sites was particularly detrimental to virus replication in the presence of MTb co-infection.
T13714 39509-39740 Sentence denotes Thus, even when three functional NF-κB binding sites are present in the LTR, as in the HIV-1 subtype C infectious clone studied here, disruption of NFAT5 binding to the LTR impairs virus replication in response to MTb co-infection.
T227 39509-39740 Sentence denotes Thus, even when three functional NF-κB binding sites are present in the LTR, as in the HIV-1 subtype C infectious clone studied here, disruption of NFAT5 binding to the LTR impairs virus replication in response to MTb co-infection.
T228 39741-39885 Sentence denotes 10.1371/journal.ppat.1002620.g006 Figure 6 Disruption of the NFAT5 site in HIV-1 subtype C significantly impairs MTb-induced viral replication.
T26297 39785-39885 Sentence denotes Disruption of the NFAT5 site in HIV-1 subtype C significantly impairs MTb-induced viral replication.
T26298 39886-39974 Sentence denotes (A) Mutation of the NFAT5 binding site in an HIV-1 subtype C infectious molecular clone.
T229 39886-39974 Sentence denotes (A) Mutation of the NFAT5 binding site in an HIV-1 subtype C infectious molecular clone.
T26299 39975-40061 Sentence denotes NFAT5 binding site mutations were introduced into the LTR of subtype C HIV-198IN22-WT.
T230 39975-40061 Sentence denotes NFAT5 binding site mutations were introduced into the LTR of subtype C HIV-198IN22-WT.
T26300 40062-40168 Sentence denotes The NFAT5 binding site mutant (HIV-198IN22 N5-Mut) LTR sequence is shown alongside that of HIV-198IN22-WT.
T231 40062-40168 Sentence denotes The NFAT5 binding site mutant (HIV-198IN22 N5-Mut) LTR sequence is shown alongside that of HIV-198IN22-WT.
T26301 40169-40256 Sentence denotes The HIV-198IN22 isolate analyzed here contains three NF-κB and two NFAT5 binding sites.
T232 40169-40256 Sentence denotes The HIV-198IN22 isolate analyzed here contains three NF-κB and two NFAT5 binding sites.
T26302 40257-40358 Sentence denotes The unique 3′ terminal adenine, which is important for NFAT5 binding to its site, is shown in blocks.
T233 40257-40358 Sentence denotes The unique 3′ terminal adenine, which is important for NFAT5 binding to its site, is shown in blocks.
T26303 40359-40441 Sentence denotes Mutations were introduced into the 3′ LTR of the HIV-198IN22-WT proviral sequence.
T234 40359-40441 Sentence denotes Mutations were introduced into the 3′ LTR of the HIV-198IN22-WT proviral sequence.
T235 40442-40542 Sentence denotes PBMC from four normal donors were infected with 1000 TCID50 of HIV-198IN22–WT or HIV-198IN22-N5-Mut.
T26304 40442-40658 Sentence denotes PBMC from four normal donors were infected with 1000 TCID50 of HIV-198IN22–WT or HIV-198IN22-N5-Mut. Cells were then (B) left infected with virus alone or (C) co-infected with MTb strain CDC1551 (10∶1 cells∶bacilli).
T236 40543-40658 Sentence denotes Cells were then (B) left infected with virus alone or (C) co-infected with MTb strain CDC1551 (10∶1 cells∶bacilli).
T26305 40659-40765 Sentence denotes Culture supernatants were collected at days 3, 7 and 11 post-infection and viral p24 levels were measured.
T237 40659-40765 Sentence denotes Culture supernatants were collected at days 3, 7 and 11 post-infection and viral p24 levels were measured.
T26306 40766-40931 Sentence denotes Replication of HIV-198IN22-N5-Mut was significantly reduced at day 11 post-infection in comparison to HIV-198IN22–WT (*, p<0.05) in the presence of MTb co-infection.
T238 40766-40931 Sentence denotes Replication of HIV-198IN22-N5-Mut was significantly reduced at day 11 post-infection in comparison to HIV-198IN22–WT (*, p<0.05) in the presence of MTb co-infection.
T239 40933-41008 Sentence denotes MTb induces NFAT5 gene expression via the MyD88-dependent signaling cascade
T14767 41009-41265 Sentence denotes To begin to decipher the cellular mechanisms underlying NFAT5-dependent, MTb-induced HIV-1 replication, we next investigated the requirement for specific signaling molecules in the TLR pathway that are required for MTb stimulation of NFAT5 gene expression.
T240 41009-41265 Sentence denotes To begin to decipher the cellular mechanisms underlying NFAT5-dependent, MTb-induced HIV-1 replication, we next investigated the requirement for specific signaling molecules in the TLR pathway that are required for MTb stimulation of NFAT5 gene expression.
T14768 41266-41352 Sentence denotes We began by examining the requirement of TLR2 ligation in enhancing NFAT5 mRNA levels.
T241 41266-41352 Sentence denotes We began by examining the requirement of TLR2 ligation in enhancing NFAT5 mRNA levels.
T14769 41353-41568 Sentence denotes When THP-1 cells were treated with the TLR2-specific agonist Pam3Cys for 16 hours, NFAT5 mRNA levels were significantly increased in Pam3Cys-stimulated cells as compared to mock-stimulated control cells (Figure 7A).
T242 41353-41568 Sentence denotes When THP-1 cells were treated with the TLR2-specific agonist Pam3Cys for 16 hours, NFAT5 mRNA levels were significantly increased in Pam3Cys-stimulated cells as compared to mock-stimulated control cells (Figure 7A).
T14770 41569-41665 Sentence denotes As a positive control for this experiment we also infected THP-1 cells with CDC1551 (Figure 7A).
T243 41569-41665 Sentence denotes As a positive control for this experiment we also infected THP-1 cells with CDC1551 (Figure 7A).
T14771 41666-41911 Sentence denotes Notably, MTb infection enhanced NFAT5 mRNA levels to a greater extent than Pam3Cys stimulation, suggesting that additional PRRs may be triggered during actual MTb infection, augmenting its stimulatory effect on NFAT5 gene expression (Figure 7A).
T244 41666-41911 Sentence denotes Notably, MTb infection enhanced NFAT5 mRNA levels to a greater extent than Pam3Cys stimulation, suggesting that additional PRRs may be triggered during actual MTb infection, augmenting its stimulatory effect on NFAT5 gene expression (Figure 7A).
T245 41912-42063 Sentence denotes 10.1371/journal.ppat.1002620.g007 Figure 7 The MyD88-mediated signaling cascade is important for MTb induction of NFAT5 expression in human monocytes.
T26959 41956-42063 Sentence denotes The MyD88-mediated signaling cascade is important for MTb induction of NFAT5 expression in human monocytes.
T26960 42064-42144 Sentence denotes (A) Engagement of TLR2 significantly enhances NFAT5 mRNA synthesis in monocytes.
T246 42064-42144 Sentence denotes (A) Engagement of TLR2 significantly enhances NFAT5 mRNA synthesis in monocytes.
T26961 42145-42293 Sentence denotes THP-1 cells were incubated with the TLR2-specific ligand Pam3Cys (5 µg/ml), infected with MTb strain CDC1551 (1∶1 cells∶bacilli), or left untreated.
T247 42145-42293 Sentence denotes THP-1 cells were incubated with the TLR2-specific ligand Pam3Cys (5 µg/ml), infected with MTb strain CDC1551 (1∶1 cells∶bacilli), or left untreated.
T26962 42294-42342 Sentence denotes After 16 hours, NFAT5 mRNA levels were measured.
T248 42294-42342 Sentence denotes After 16 hours, NFAT5 mRNA levels were measured.
T26963 42343-42568 Sentence denotes NFAT5 mRNA expression was significantly increased when the cells were incubated with Pam3Cys (*, p<0.05) or infected with MTb (**, p<0.01). (B) MyD88 is important for MTb induction of NFAT5 gene expression in human monocytes.
T249 42343-42568 Sentence denotes NFAT5 mRNA expression was significantly increased when the cells were incubated with Pam3Cys (*, p<0.05) or infected with MTb (**, p<0.01). (B) MyD88 is important for MTb induction of NFAT5 gene expression in human monocytes.
T250 42569-42740 Sentence denotes THP-1 cells that constitutively express lentivirally-delivered shRNA targeting MyD88 or control shRNA targeting GFP were constructed as described in Materials and Methods.
T26964 42569-42867 Sentence denotes THP-1 cells that constitutively express lentivirally-delivered shRNA targeting MyD88 or control shRNA targeting GFP were constructed as described in Materials and Methods. Cells were infected with MTb strain CDC1551 (1∶1 cells∶bacilli) or left uninfected for 16 hours at 37°C, and RNA was analyzed.
T251 42741-42867 Sentence denotes Cells were infected with MTb strain CDC1551 (1∶1 cells∶bacilli) or left uninfected for 16 hours at 37°C, and RNA was analyzed.
T26965 42868-43094 Sentence denotes NFAT5 mRNA levels were significantly inhibited in the cells transduced with MyD88-specific shRNA (black bars) compared with the cells transduced with control shRNA (open bars) in both, the absence or presence of MTb infection.
T252 42868-43094 Sentence denotes NFAT5 mRNA levels were significantly inhibited in the cells transduced with MyD88-specific shRNA (black bars) compared with the cells transduced with control shRNA (open bars) in both, the absence or presence of MTb infection.
T26966 43095-43338 Sentence denotes TNF and CD86 mRNAs, the expression of which are MyD88-dependent and -independent, respectively, during MTb infection, were measured as controls. (C, D) IRAK1 and TRAF6 are required for MTb induction of NFAT5 gene expression in human monocytes.
T253 43095-43338 Sentence denotes TNF and CD86 mRNAs, the expression of which are MyD88-dependent and -independent, respectively, during MTb infection, were measured as controls. (C, D) IRAK1 and TRAF6 are required for MTb induction of NFAT5 gene expression in human monocytes.
T254 43339-43488 Sentence denotes THP-1 cells that constitutively express lentivirally-delivered IRAK1- or TRAF6-specific shRNA were constructed as described in Materials and Methods.
T26967 43339-43555 Sentence denotes THP-1 cells that constitutively express lentivirally-delivered IRAK1- or TRAF6-specific shRNA were constructed as described in Materials and Methods. THP-1 cells expressing control shRNA were used as described in 7B.
T255 43489-43555 Sentence denotes THP-1 cells expressing control shRNA were used as described in 7B.
T26968 43556-43662 Sentence denotes Cells were infected with MTb strain CDC1551 or left uninfected for 16 hours at 37°C, and RNA was analyzed.
T256 43556-43662 Sentence denotes Cells were infected with MTb strain CDC1551 or left uninfected for 16 hours at 37°C, and RNA was analyzed.
T26969 43663-43849 Sentence denotes NFAT5 expression was significantly inhibited in the MTb-infected cells expressing either IRAK1 (C) or TRAF6 (D) shRNA (black bars) compared to cells expressing control shRNA (open bars).
T257 43663-43849 Sentence denotes NFAT5 expression was significantly inhibited in the MTb-infected cells expressing either IRAK1 (C) or TRAF6 (D) shRNA (black bars) compared to cells expressing control shRNA (open bars).
T26970 43850-43992 Sentence denotes TNF and CD86 mRNAs, both of which require IRAK1 and TRAF6 to be induced by PRR activation, were measured as controls. (*, p<0.05; **, p<0.01).
T258 43850-43992 Sentence denotes TNF and CD86 mRNAs, both of which require IRAK1 and TRAF6 to be induced by PRR activation, were measured as controls. (*, p<0.05; **, p<0.01).
T14772 43993-44076 Sentence denotes The adaptor molecule MyD88 transmits signals upon TLR2 ligation (reviewed in [53]).
T259 43993-44076 Sentence denotes The adaptor molecule MyD88 transmits signals upon TLR2 ligation (reviewed in [53]).
T14773 44077-44337 Sentence denotes To confirm that this protein and, therefore, that this TLR-dependent pathway is important for MTb induction of NFAT5 gene expression, we constructed a THP-1 cell line that constitutively expresses a lentivirally-delivered short hairpin (sh)RNA targeting MyD88.
T260 44077-44337 Sentence denotes To confirm that this protein and, therefore, that this TLR-dependent pathway is important for MTb induction of NFAT5 gene expression, we constructed a THP-1 cell line that constitutively expresses a lentivirally-delivered short hairpin (sh)RNA targeting MyD88.
T14774 44338-44570 Sentence denotes Real-time PCR analysis demonstrated that MyD88 mRNA levels in these cells were constitutively and significantly reduced in comparison to control THP-1 cells transduced with a lentivirus expressing control GFP shRNA (data not shown).
T261 44338-44570 Sentence denotes Real-time PCR analysis demonstrated that MyD88 mRNA levels in these cells were constitutively and significantly reduced in comparison to control THP-1 cells transduced with a lentivirus expressing control GFP shRNA (data not shown).
T14775 44571-44679 Sentence denotes These MyD88 knockdown THP-1 cells were then infected with MTb (CDC1551) and NFAT5 mRNA levels were examined.
T262 44571-44679 Sentence denotes These MyD88 knockdown THP-1 cells were then infected with MTb (CDC1551) and NFAT5 mRNA levels were examined.
T14776 44680-44784 Sentence denotes Since TNF is a MyD88-dependent gene [21], [54], [55], as a positive control we measured TNF mRNA levels.
T263 44680-44784 Sentence denotes Since TNF is a MyD88-dependent gene [21], [54], [55], as a positive control we measured TNF mRNA levels.
T14777 44785-44879 Sentence denotes For a negative control, we measured mRNA levels of the MyD88-independent gene CD86 [55], [56].
T264 44785-44879 Sentence denotes For a negative control, we measured mRNA levels of the MyD88-independent gene CD86 [55], [56].
T14778 44880-45021 Sentence denotes As shown in Figure 7B, knockdown of MyD88 specifically inhibited MTb-induced NFAT5 and TNF mRNA levels and had no impact on CD86 mRNA levels.
T265 44880-45021 Sentence denotes As shown in Figure 7B, knockdown of MyD88 specifically inhibited MTb-induced NFAT5 and TNF mRNA levels and had no impact on CD86 mRNA levels.
T14779 45022-45076 Sentence denotes Thus, MTb regulation of NFAT5 is dependent upon MyD88.
T266 45022-45076 Sentence denotes Thus, MTb regulation of NFAT5 is dependent upon MyD88.
T14780 45077-45306 Sentence denotes To futher dissect the pathway mediating MTb upregulation of NFAT5 transcription, we also constructed THP-1 cells transduced with lentivirally-delivered shRNAs targeting IRAK1 or TRAF6, two signaling molecules downstream of MyD88.
T267 45077-45306 Sentence denotes To futher dissect the pathway mediating MTb upregulation of NFAT5 transcription, we also constructed THP-1 cells transduced with lentivirally-delivered shRNAs targeting IRAK1 or TRAF6, two signaling molecules downstream of MyD88.
T14781 45307-45426 Sentence denotes As shown in Figures 7C and 7D, knockdown of both IRAK1 and TRAF6 significantly inhibited MTb induced NFAT5 mRNA levels.
T268 45307-45426 Sentence denotes As shown in Figures 7C and 7D, knockdown of both IRAK1 and TRAF6 significantly inhibited MTb induced NFAT5 mRNA levels.
T14782 45427-45636 Sentence denotes As expected, MTb-induced TNF gene expression was also significantly reduced (Figures 7C–7D), consistent with a role for IRAK1 and TRAF6 in TNF gene expression secondary to MTb infection as previously reported.
T269 45427-45636 Sentence denotes As expected, MTb-induced TNF gene expression was also significantly reduced (Figures 7C–7D), consistent with a role for IRAK1 and TRAF6 in TNF gene expression secondary to MTb infection as previously reported.
T14783 45637-45917 Sentence denotes Although CD86 gene expression did not depend on MyD88 (Figure 7B), abrogation of IRAK1 and TRAF6 gene expression significantly reduced CD86 mRNA levels, indicating that CD86 expression is IRAK1- and TRAF6-dependent, but MyD88-independent in MTb-infected monocytes (Figures 7C–7D).
T270 45637-45917 Sentence denotes Although CD86 gene expression did not depend on MyD88 (Figure 7B), abrogation of IRAK1 and TRAF6 gene expression significantly reduced CD86 mRNA levels, indicating that CD86 expression is IRAK1- and TRAF6-dependent, but MyD88-independent in MTb-infected monocytes (Figures 7C–7D).
T14784 45918-46128 Sentence denotes Taken together, these results clearly demonstrate that MTb upregulation of NFAT5 gene expression in human monocytic cells requires activation of MyD88 and the MyD88-associated adaptor molecules IRAK1 and TRAF6.
T271 45918-46128 Sentence denotes Taken together, these results clearly demonstrate that MTb upregulation of NFAT5 gene expression in human monocytic cells requires activation of MyD88 and the MyD88-associated adaptor molecules IRAK1 and TRAF6.
T272 46130-46140 Sentence denotes Discussion
T17206 46141-46256 Sentence denotes The transcription factor NFAT5 is the most evolutionarily divergent member of the Rel family of NFAT proteins [57].
T273 46141-46256 Sentence denotes The transcription factor NFAT5 is the most evolutionarily divergent member of the Rel family of NFAT proteins [57].
T17207 46257-46568 Sentence denotes Unlike the other NFAT proteins (NFATp, NFATc, NFAT3, and NFAT4), NFAT5 binds DNA as an obligate dimer in a fashion resembling NF-κB proteins [58], is calcineurin independent, and does not cooperate with the basic region-leucine zipper proteins Fos and Jun in gene activation ([59], [60], reviewed in [61]–[63]).
T274 46257-46568 Sentence denotes Unlike the other NFAT proteins (NFATp, NFATc, NFAT3, and NFAT4), NFAT5 binds DNA as an obligate dimer in a fashion resembling NF-κB proteins [58], is calcineurin independent, and does not cooperate with the basic region-leucine zipper proteins Fos and Jun in gene activation ([59], [60], reviewed in [61]–[63]).
T17208 46569-46698 Sentence denotes To date, osmotic stress, integrin activation, and T cell-stimulation have been shown to regulate NFAT5 activity [59], [64], [65].
T275 46569-46698 Sentence denotes To date, osmotic stress, integrin activation, and T cell-stimulation have been shown to regulate NFAT5 activity [59], [64], [65].
T17209 46699-46916 Sentence denotes Based on our demonstration here that the host innate immune response to MTb infection induces MyD88-dependent upregulation of NFAT5 gene expression, we have expanded this list to include MTb as an additional stimulus.
T276 46699-46916 Sentence denotes Based on our demonstration here that the host innate immune response to MTb infection induces MyD88-dependent upregulation of NFAT5 gene expression, we have expanded this list to include MTb as an additional stimulus.
T17210 46917-47122 Sentence denotes We have linked this observation to MTb-stimulated HIV-1 gene expression by showing that MTb enhances the replication of HIV-1 subtypes B and C via a direct interaction between NFAT5 and the viral promoter.
T277 46917-47122 Sentence denotes We have linked this observation to MTb-stimulated HIV-1 gene expression by showing that MTb enhances the replication of HIV-1 subtypes B and C via a direct interaction between NFAT5 and the viral promoter.
T17211 47123-47262 Sentence denotes Thus, HIV-1 has co-opted NFAT5, which is induced as part of the innate immune response to TB, for enhanced viral transcription/replication.
T278 47123-47262 Sentence denotes Thus, HIV-1 has co-opted NFAT5, which is induced as part of the innate immune response to TB, for enhanced viral transcription/replication.
T17212 47263-47322 Sentence denotes NF-κB is activated after MTb engagement of PRRs [20], [66].
T279 47263-47322 Sentence denotes NF-κB is activated after MTb engagement of PRRs [20], [66].
T17213 47323-47530 Sentence denotes Multiple studies of HIV-1 activation have shown that initial recruitment of the NF-κB p50/p65 heterodimer to the HIV-1 proviral enhancer region is crucial for HIV gene transcription (reviewed in [67], [68]).
T280 47323-47530 Sentence denotes Multiple studies of HIV-1 activation have shown that initial recruitment of the NF-κB p50/p65 heterodimer to the HIV-1 proviral enhancer region is crucial for HIV gene transcription (reviewed in [67], [68]).
T17214 47531-47819 Sentence denotes Upon interaction with the viral LTR, NF-κB rapidly induces hyperacetylation of histones associated with nucleosome 1 (nuc-1) at the HIV-1 transcription start site, resulting in recruitment of the pTEFb complex, which is required for RNA pol II processivity ([69], reviewed in [68], [70]).
T281 47531-47819 Sentence denotes Upon interaction with the viral LTR, NF-κB rapidly induces hyperacetylation of histones associated with nucleosome 1 (nuc-1) at the HIV-1 transcription start site, resulting in recruitment of the pTEFb complex, which is required for RNA pol II processivity ([69], reviewed in [68], [70]).
T17215 47820-48021 Sentence denotes In this report we directly examined the relative roles of NFAT5 and NF-κB p50/p65 in HIV-1 replication under conditions of activation by MTb co-infection, when NF-κB levels in the nucleus are elevated.
T282 47820-48021 Sentence denotes In this report we directly examined the relative roles of NFAT5 and NF-κB p50/p65 in HIV-1 replication under conditions of activation by MTb co-infection, when NF-κB levels in the nucleus are elevated.
T17216 48022-48247 Sentence denotes We found that specific disruption of the NFAT5 binding site(s) in R5-tropic subtype B or subtype C infectious molecular clones significantly reduced virus replication in PBMC or MDM co-infected with a clinical isolate of MTb.
T283 48022-48247 Sentence denotes We found that specific disruption of the NFAT5 binding site(s) in R5-tropic subtype B or subtype C infectious molecular clones significantly reduced virus replication in PBMC or MDM co-infected with a clinical isolate of MTb.
T17217 48248-48395 Sentence denotes Thus, NF-κB binding to the viral LTR is not sufficient to compensate for the loss of NFAT5 binding to the LTR under conditions of MTb co-infection.
T284 48248-48395 Sentence denotes Thus, NF-κB binding to the viral LTR is not sufficient to compensate for the loss of NFAT5 binding to the LTR under conditions of MTb co-infection.
T17218 48396-48490 Sentence denotes Reciprocally, an intact NFAT5 site could not compensate for disruption of the two NF-κB sites.
T285 48396-48490 Sentence denotes Reciprocally, an intact NFAT5 site could not compensate for disruption of the two NF-κB sites.
T17219 48491-48557 Sentence denotes Thus, both factors are required for MTb-induced HIV-1 replication.
T286 48491-48557 Sentence denotes Thus, both factors are required for MTb-induced HIV-1 replication.
T287 48558-48752 Sentence denotes Intriguingly, both in the absence and presence of MTb co-infection, disruption of NF-κB site II consistently resulted in greater suppression of virus replication than disruption of NF-κB site I.
T17220 48558-48899 Sentence denotes Intriguingly, both in the absence and presence of MTb co-infection, disruption of NF-κB site II consistently resulted in greater suppression of virus replication than disruption of NF-κB site I. Consistent with these findings, synthetic reporter assays have previously shown that these two sites have distinct roles in driving transcription.
T288 48753-48899 Sentence denotes Consistent with these findings, synthetic reporter assays have previously shown that these two sites have distinct roles in driving transcription.
T17221 48900-49076 Sentence denotes The downstream NF-κB site I binding site, which is directly upstream of three highly conserved Sp binding sites, appears to enhance Sp protein recruitment to the LTR [71]–[73].
T289 48900-49076 Sentence denotes The downstream NF-κB site I binding site, which is directly upstream of three highly conserved Sp binding sites, appears to enhance Sp protein recruitment to the LTR [71]–[73].
T17222 49077-49265 Sentence denotes Because NF-κB site I overlaps the NFAT5 binding site, it is possible that loss of NF-κB binding to this site is mitigated, at least in part, by increased NFAT5 binding to the mutated site.
T290 49077-49265 Sentence denotes Because NF-κB site I overlaps the NFAT5 binding site, it is possible that loss of NF-κB binding to this site is mitigated, at least in part, by increased NFAT5 binding to the mutated site.
T17223 49266-49448 Sentence denotes This hypothesis is consistent with our quantitative DNase I footprinting analysis that shows that specific disruption of NF-κB site I results in enhanced binding of NFAT5 to the LTR.
T291 49266-49448 Sentence denotes This hypothesis is consistent with our quantitative DNase I footprinting analysis that shows that specific disruption of NF-κB site I results in enhanced binding of NFAT5 to the LTR.
T17224 49449-49695 Sentence denotes HIV-1 subtype C now makes up greater than 50% of all HIV-1 infections worldwide, and its prevalence is especially high in regions of endemic TB [1], [2], [28], [52], Most HIV-1 subtype C isolates possess three NF-κB binding sites in the LTR [30].
T292 49449-49695 Sentence denotes HIV-1 subtype C now makes up greater than 50% of all HIV-1 infections worldwide, and its prevalence is especially high in regions of endemic TB [1], [2], [28], [52], Most HIV-1 subtype C isolates possess three NF-κB binding sites in the LTR [30].
T17225 49696-49970 Sentence denotes Our analysis of MTb whole lysate-stimulated, LTR-driven reporter gene expression from representative B, C, and E LTRs showed that LTRs derived from subtype C isolates were superior in driving reporter gene expression as compared to LTRs derived from subtype B or E isolates.
T293 49696-49970 Sentence denotes Our analysis of MTb whole lysate-stimulated, LTR-driven reporter gene expression from representative B, C, and E LTRs showed that LTRs derived from subtype C isolates were superior in driving reporter gene expression as compared to LTRs derived from subtype B or E isolates.
T17226 49971-50120 Sentence denotes Other studies have found that, in response to TNF, subtype C LTRs drive reporter gene transcription more strongly than other subtype LTRs [38], [39].
T294 49971-50120 Sentence denotes Other studies have found that, in response to TNF, subtype C LTRs drive reporter gene transcription more strongly than other subtype LTRs [38], [39].
T17227 50121-50649 Sentence denotes In the co-infection model utilized in this report, in which clinical isolates of both MTb and HIV-1 subtype C were used to infect freshly prepared human peripheral blood cells, we found that specific disruption of NFAT5 binding to the LTR significantly impaired viral replication during MTb co-infection, indicating that even the presence of three intact NF-κB binding sites, which is typical for HIV-1 subtype C isolates [30], cannot compensate for loss of NFAT5 recruitment to the viral LTR in the context of MTb co-infection.
T295 50121-50649 Sentence denotes In the co-infection model utilized in this report, in which clinical isolates of both MTb and HIV-1 subtype C were used to infect freshly prepared human peripheral blood cells, we found that specific disruption of NFAT5 binding to the LTR significantly impaired viral replication during MTb co-infection, indicating that even the presence of three intact NF-κB binding sites, which is typical for HIV-1 subtype C isolates [30], cannot compensate for loss of NFAT5 recruitment to the viral LTR in the context of MTb co-infection.
T17228 50650-50841 Sentence denotes Given that the NF-κB and NFAT5 binding motifs overlap, how do NF-κB and NFAT5 function at this overlapping or shared site in the LTR to drive viral transcription in response to MTb infection?
T296 50650-50841 Sentence denotes Given that the NF-κB and NFAT5 binding motifs overlap, how do NF-κB and NFAT5 function at this overlapping or shared site in the LTR to drive viral transcription in response to MTb infection?
T17229 50842-51062 Sentence denotes Because NFAT5 expression continues to rise for at least 48 hours post-MTb infection (Figure 2A), one possibility is that the host factors NF-κB and NFAT5 associate with the LTR at different stages after viral activation.
T297 50842-51062 Sentence denotes Because NFAT5 expression continues to rise for at least 48 hours post-MTb infection (Figure 2A), one possibility is that the host factors NF-κB and NFAT5 associate with the LTR at different stages after viral activation.
T17230 51063-51174 Sentence denotes While NF-κB presence in the nucleus declines in the hours post MTb infection [74], [75], NFAT5 levels escalate.
T298 51063-51174 Sentence denotes While NF-κB presence in the nucleus declines in the hours post MTb infection [74], [75], NFAT5 levels escalate.
T17231 51175-51431 Sentence denotes Thus, NFAT5, which is pivotal for HIV-1 transcription in unstimulated cells [31], may, after Mtb infection, perpetuate viral transcriptional initiation begun by NF-κB and, with Tat levels also elevated, promote high levels of sustained HIV-1 transcription.
T299 51175-51431 Sentence denotes Thus, NFAT5, which is pivotal for HIV-1 transcription in unstimulated cells [31], may, after Mtb infection, perpetuate viral transcriptional initiation begun by NF-κB and, with Tat levels also elevated, promote high levels of sustained HIV-1 transcription.
T17232 51432-51586 Sentence denotes Indeed, in response to hypertonic stress, NFAT5 binds to the aldose reductase (AR) promoter and induces rapid hyperacetylation of histones H3 and H4 [76].
T300 51432-51586 Sentence denotes Indeed, in response to hypertonic stress, NFAT5 binds to the aldose reductase (AR) promoter and induces rapid hyperacetylation of histones H3 and H4 [76].
T17233 51587-51750 Sentence denotes Furthermore, hypertonic stress of mouse renal collecting duct epithelial cells results in NFAT5 and NF-κB complex formation at NF-κB-dependent gene promoters [77].
T301 51587-51750 Sentence denotes Furthermore, hypertonic stress of mouse renal collecting duct epithelial cells results in NFAT5 and NF-κB complex formation at NF-κB-dependent gene promoters [77].
T17234 51751-51988 Sentence denotes Thus, there are precedents for imagining that NFAT5 could function at the HIV-1 LTR by inducing histone remodeling in a manner similar to NF-κB ([69], reviewed in [68], [70]) or by directly interacting with NF-κB in a cooperative manner.
T302 51751-51988 Sentence denotes Thus, there are precedents for imagining that NFAT5 could function at the HIV-1 LTR by inducing histone remodeling in a manner similar to NF-κB ([69], reviewed in [68], [70]) or by directly interacting with NF-κB in a cooperative manner.
T17235 51989-52174 Sentence denotes In conclusion, we have demonstrated that NFAT5 is required for the replication of R5-tropic subtype B and subtype C HIV-1 isolates in response to MTb-co-infection of human PBMC and MDM.
T303 51989-52174 Sentence denotes In conclusion, we have demonstrated that NFAT5 is required for the replication of R5-tropic subtype B and subtype C HIV-1 isolates in response to MTb-co-infection of human PBMC and MDM.
T17236 52175-52362 Sentence denotes Functional NF-κB interaction with the viral LTR is also required, but fully intact NF-κB binding elements are unable to compensate for the loss of NFAT5 recruitment to the viral promoter.
T304 52175-52362 Sentence denotes Functional NF-κB interaction with the viral LTR is also required, but fully intact NF-κB binding elements are unable to compensate for the loss of NFAT5 recruitment to the viral promoter.
T17237 52363-52510 Sentence denotes In addition, we have demonstrated that MTb infection or stimulation with the TLR2 ligand PAM3Cys, induces NFAT5 gene expression in human monocytes.
T305 52363-52510 Sentence denotes In addition, we have demonstrated that MTb infection or stimulation with the TLR2 ligand PAM3Cys, induces NFAT5 gene expression in human monocytes.
T17238 52511-52646 Sentence denotes Furthermore, we have shown that MTb stimulation of NFAT5 depends on TLR pathway signaling molecules, including MyD88, IRAK1, and TRAF6.
T306 52511-52646 Sentence denotes Furthermore, we have shown that MTb stimulation of NFAT5 depends on TLR pathway signaling molecules, including MyD88, IRAK1, and TRAF6.
T17239 52647-52942 Sentence denotes Taken together, the findings presented here enhance the general understanding of the innate immune response to MTb infection by showing that NFAT5 is a major mediator of TLR-dependent gene expression; its importance for gene regulation is likely applicable to other MTb- and TLR-regulated genes.
T307 52647-52942 Sentence denotes Taken together, the findings presented here enhance the general understanding of the innate immune response to MTb infection by showing that NFAT5 is a major mediator of TLR-dependent gene expression; its importance for gene regulation is likely applicable to other MTb- and TLR-regulated genes.
T17240 52943-53148 Sentence denotes Moreover, these data provide molecular insights into MTb regulation of HIV-1 transcription, thereby elucidating several new targets for therapeutic interventions aimed at controlling TB/HIV-1 co-infection.
T308 52943-53148 Sentence denotes Moreover, these data provide molecular insights into MTb regulation of HIV-1 transcription, thereby elucidating several new targets for therapeutic interventions aimed at controlling TB/HIV-1 co-infection.